Complementing cell lines

ABSTRACT

A packaging cell line capable of complemention recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably human embryonic kidney cells and primary human amniocytes) which are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/573,740, filed May 18, 2000, pending, which claims the benefit of thefiling date of U.S. Provisional Application Serial No. 60/134,764 filedMay 18, 1999.

TECHNICAL FIELD

The invention relates to the field of biotechnology generally, and morespecifically to adenoviral-based complementing cell lines.

BACKGROUND

Typically, vector and packaging cells have to be adapted to one anotherso that they have all the necessary elements, but they do not haveoverlapping elements which lead to replication competent virus byrecombination. Therefore, the sequences necessary for propertranscription of the packaging construct may be heterologous regulatorysequences derived from, for example, other human adenovirus (Ad)serotypes, non-human adenoviruses, other viruses like, but not limitedto, SV40, hepatitis B virus (HBV), Rous Sarcoma Virus (RSV), cytomegalovirus (CMV), etc. or from higher eukaryotes such as mammals. In general,these sequences include a promoter, enhancer and polyadenylationsequences.

PER.C6 (ECACC deposit number 96022940) is an example of a cell linedevoid of sequence overlap between the packaging construct and theadenoviral vector (Fallaux et al., 1998). Recombinant viruses based onsubgroup C adenoviruses such as Ad5 and Ad2 can be propagatedefficiently on these packaging cells. Generation and propagation ofadenoviruses from other serotypes, like subgroup B viruses, has provento be more difficult on PER.C6 cells. However, as described in Europeanpatent application 00201738.2, recombinant viruses based on subgroup Bvirus Ad35 can be made by co-transfection of an expression constructcontaining the Ad35 early region-1 sequences (Ad35-E1). Furthermore,Ad35-based viruses that are deleted for E1A sequences were shown toreplicate efficiently on PER.C6 cells. Thus, the E1A proteins of Ad5complement Ad35-E1A functions, whereas at least part of the E1Bfunctions of Ad35 are necessary. This serotype specificity in E1Bfunctions was recently also described for Ad7 recombinant viruses. In anattempt to generate recombinant adenoviruses derived from subgroup Bvirus Ad7, Abrahamsen et al. (1997) were not able to generate E1-deletedviruses on 293 cells without contamination of wild-type (wt) Ad7.Viruses that were picked after plaque purification on 293-ORF6 cells(Brough et al., 1996) were shown to have incorporated Ad7 E1B sequencesby non-homologous recombination. Thus, efficient propagation of Ad7recombinant viruses proved possible only in the presence of Ad7-E1Bexpression and Ad5-E4-ORF6 expression. The E1B proteins are known tointeract with cellular as well as viral proteins (Bridge et al., 1993;White, 1995). Possibly, the complex formed between the E1B 55K proteinand E4-ORF6 which is necessary to increase MRNA export of viral proteinsand to inhibit export of most cellular mRNAs, is critical and in someway serotype specific.

DESCRIPTION OF THE INVENTION

The present invention provides new packaging cell lines capable ofcomplementing recombinant adenoviruses based on serotypes other thansubgroup C viruses, such as serotypes from subgroup B, like adenovirustype 35.

In one aspect of the invention, the new packaging cells are derived fromprimary, diploid human cells such as, but not limited to, primary humanretinoblasts, primary human embryonic kidney cells or primary humanamniocytes. Transfection of primary cells with the adenovirus E1A genealone can induce unlimited proliferation (immortalisation), but does notresult in complete transformation. However, expression of E1A in mostcases results in induction of programmed cell death (apoptosis), andoccasionally immortalisation is obtained (Jochemsen et al., 1987).Co-expression of the E1B gene is required to prevent induction ofapoptosis and for complete morphological transformation to occur(reviewed in White, 1995). Therefore, in one aspect of the invention,primary human cells are transformed by expression of adenovirus E1proteins of a subgroup other than subgroup C, preferably subgroup B,more preferably adenovirus type 35. The combined activity of the E1A andE1B proteins establishes indefinite growth of the cells and enablescomplementation of recombinant adenoviruses.

In another aspect of the invention, the transforming E1 sequences arederived from different serotypes. As disclosed in European Patentapplication 00201738.2, Ad35 E1 sequences are capable of transformingBaby Rat Kidney (BRK) cells, albeit with a lower efficiency than thatseen with Ad5 E1 sequences. This was also observed for E1 sequences fromAd12 (Bernards et al., 1982). Therefore, in this aspect of theinvention, primary diploid human cells are transformed with chimeric E1constructs that consist of part of the E1 sequences of a serotype thatenables efficient transformation of primary human cells and part of theE1 sequences of another serotype which E1 sequences provide theserotype-specific E1B function(s) that enable(s) efficient propagationof E1-deleted viruses of that serotype. In a preferred embodiment ofthis aspect of the invention, the E1A region is derived from a subgroupC adenovirus, like, but not limited to, Ad5, and the E1B sequences arederived from an alternative adenovirus more particularly from anadenovirus of subgroup B, more particularly from adenovirus type 35. Ina more preferred embodiment, the E1A sequences and the E1B-21K sequencesare derived from a subgroup C adenovirus, like, but not limited to, Ad5,and the E1B-55k sequences as far as not overlapping with the 21Ksequences are derived from an adenovirus of subgroup B, more particularfrom adenovirus type 35. In an even more preferred embodiment, all E1sequences are derived from a subgroup C adenovirus, like but not limitedto Ad5, except for at least the part of the E1B-55K sequences that arenecessary for serotype-specific complementation of an alternativeadenovirus subgroup, more particular adenovirus subgroup B, moreparticular adenovirus type 35, the sequences being derived from theadenovirus.

The primary diploid human cells are transformed by adenovirus E1sequences either operatively linked on one DNA molecule or located ontwo separate DNA molecules. In the latter case, one DNA molecule carriesat least part of the E1 sequences of the serotype enabling efficienttransformation and the second DNA molecule carries at least part of thesequences necessary for serotype-specific complementation. In allaspects, the sequences are operatively linked to regulatory sequencesenabling transcription and translation of the encoded proteins.

In another aspect of the invention, new packaging cells are describedthat are derived from PER.C6 (ECACC deposit number 96022940; Fallaux etal., 1998) and contain Ad35-E1 sequences integrated into their genome.These Ad35-E1 sequences are present in a functional expression cassette,but preferably do not contain sequences overlapping with sequencespresent in the recombinant viral vector. Preferably, the functionalexpression cassette consists of a heterologous promoter andpoly-adenylation signal functionally linked to Ad35-E1 sequences. Morespecifically, the Ad35-E1 sequences are functionally linked to the humanphosphoglycerate gene promoter (hPGK) and hepatitis B viruspoly-adenylation signal (HBV-pA). Preferably, Ad35-E1 sequences comprisethe coding regions of the E1A proteins and the E1B promoter sequenceslinked to E1B coding sequences up to and including the stop codon of theE1B 55K protein. More preferably, the Ad35-E1 sequences comprisenucleotide 468 to nucleotide 3400 of the Ad35 wt sequence. To be able toselect for transfected cells, a dominant selection marker like, but notlimited to, the neor gene has to be incorporated on the expressionvector or the Ad35 expression vector is co-transfected with a separateexpression vector mediating expression of the selection marker. In bothcases, the selection marker becomes integrated in the cellular genome.Other AdS-E1 transformed cell lines like 293 (Graham et al., 1977) and911 (Fallaux et al., 1996) or established human cell lines like A549cells may be used without departing from the present invention.

In another aspect of the invention, PER.C6-derived cells are describedthat express functional Ad35 E1B sequences. In one embodiment, theAd35-E1B sequences are driven by the E1B promoter and terminated by aheterologous poly-adenylation signal like, but not limited to, theHBVpA. In a preferred embodiment, the Ad35-E1B sequences are driven by aheterologous promoter like, but not limited to, the hPGK promoter orElongation Factor-1α (EF-1α) promoter and terminated by a heterologouspA signal like, but not limited to, the HBVpA. These Ad35-E1B sequencespreferably comprise the coding regions of the E1B 21K and the E1B 55Kproteins located between nucleotides 1611 and 3400 of the wild-type (wt)Ad35 sequence. More preferably, the Ad35-E1B sequences comprisenucleotides 1550 to 3400 of the wt Ad35 sequence. In an even morepreferred embodiment, the E1B sequences comprise the coding sequences ofthe E1B-55K gene located between nucleotides 1916 and 3400 of the wtAd35 sequence.

Cell lines subject of this invention are useful for, among other things,the production of recombinant adenoviruses designed for gene therapy andvaccination. The cell lines, being derived from cells of human origin,are also useful for the production of human recombinant therapeuticproteins like, but not limited to human growth factors, humanantibodies. In addition the cell lines are useful for the production ofhuman viruses other than adenovirus like, but not limited to, influenzavirus, herpes simplex virus, rotavirus, measles virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Bar graph showing the percentage of serum samples positive forneutralization for each human wt adenovirus tested (see, Example fordescription of the neutralization assay).

FIG. 2: Graph showing absence of correlation between the VP/CCID50 ratioand the percentage of neutralization.

FIG. 3: Bar graph presenting the percentage sera samples that showneutralizing activity to a selection of adenovirus serotypes. Sera werederived from healthy volunteers from Belgium and the UK.

FIG. 4: Bar graph presenting the percentage sera samples that showneutralizing activity to adenovirus serotypes 5, 11, 26, 34, 35, 48 and49. Sera were derived from five different locations in Europe and theUnited States.

FIG. 5: Sequence of human adenovirus type 35.

FIG. 6: Map of pAdApt35IP1.

FIG. 7: Schematic representation of the steps undertaken to constructpWE.Ad35.pIX-rITR.

FIG. 8: Map of pWE.Ad35.pIX-rITR.

FIG. 9: Map of pRSV.Ad35-E1.

FIG. 10: Map of PGKneopA.

FIG. 11: Map of pRSVpNeo.

FIG. 12: Map of pRSVhbvNeo.

FIG. 13: Map of pIG.E1A.E1B.

FIG. 14: Map of pIG135.

FIG. 15: Map of pIG270.

FIG. 16: Map of pBr.Ad35.leftITR-pIX.

FIG. 17: Map of pBr.Ad35.leftITR-pIXΔE1A

FIG. 18: Map of pBr.Ad35.Δ21K

FIG. 19: Map of pBr.Ad35.Δ55K1

FIG. 20: Map of pBrAd35ΔSM

FIG. 21: Schematic representation of Ad35-E1A/E1B deletion constructs.

FIG. 22: Map of pIG.35BL.

FIG. 23: Map of pRSVneo4.

FIG. 24: Map of pIG35Bneo.

FIG. 25: Map of pIG35.55K

FIG. 26: Map of pIG535

FIG. 27: Map of pIG635

FIG. 28: Map of pIG735

DETAILED DESCRIPTION OF THE INVENTION

The invention is further explained by the use of the followingillustrative examples.

EXAMPLES Example 1 A High Throughput Assay for the Detection ofNeutralizing Activity in Human Serum

To enable screening of a large amount of human sera for the presence ofneutralizing antibodies against all adenovirus serotypes, an automated96-wells assay was developed.

Human Sera

A panel of 100 individuals was selected. Volunteers (50% male, 50%female) were healthy individuals between ages 20 and 60 years old withno restriction for race. All volunteers signed an informed consent form.People professionally involved in adenovirus research were excluded.

Approximately 60 ml blood was drawn in dry tubes. Within two hours aftersampling, the blood was centrifuged at 2500 rpm for 10 minutes.Approximately 30 ml serum was transferred to polypropylene tubes andstored frozen at −20° C. until further

Serum was thawed and heat-inactivated at 56° C. for 10 minutes and thenaliquoted to prevent repeated cycles of freeze/thawing. Part was used tomake five steps of twofold dilutions in medium (DMEM, Gibco BRL) in aquantity enough to fill out approximately 70 96-well plates. Aliquots ofundiluted and diluted sera were pipetted in deep well plates (96-wellformat) and using a programmed platemate dispensed in 100 μl aliquotsinto 96-well plates. This way the plates were loaded with eightdifferent sera in (100 μl/well) according to the scheme below:

S1/ S1/ S1/ S1/ S1/ S5/ S5/ S5/ S5/ S5/ — — 2 4 8 16 32 2 4 8 16 32 S1/S1/ S1/ S1/ S1/ S5/ S5/ S5/ S5/ S5/ — — 2 4 8 16 32 2 4 8 16 32 S2/ S2/S2/ S2/ S2/ S6/ S6/ S6/ S6/ S6/ — — 2 4 8 16 32 2 4 8 16 32 S2/ S2/ S2/S2/ S2/ S6/ S6/ S6/ S6/ S6/ — — 2 4 8 16 32 2 4 8 16 32 S3/ S3/ S3/ S3/S3/ S7/ S7/ S7/ S7/ S7/ — — 2 4 8 16 32 2 4 8 16 32 S3/ S3/ S3/ S3/ S3/S7/ S7/ S7/ S7/ S7/ — — 2 4 8 16 32 2 4 8 16 32 S4/ S4/ S3/ S3/ S3/ S8/S8/ S8/ S8/ S8/ — — 2 4 8 16 32 2 4 8 16 32 S4/ S4/ S3/ S3/ S3/ S8/ S8/S8/ S8/ S8/ — — 2 4 8 16 32 2 4 8 16 32

Where S1/2 to S8/2 in columns 1 and 6 represent 1× diluted sera andSx/4, Sx/8, Sx/16 and Sx/32 the twofold serial dilutions. The lastplates also contained four wells filled with 100 μl fetal calf serum asa negative control. Plates were kept at −20° C. until further use.

Preparation of Human Adenovirus Stocks

Prototypes of all known human adenoviruses were inoculated on T25 flasksseeded with PER.C6 cells (Fallaux et al., 1998) and harvested upon fullCPE. After freeze/thawing 1-2 ml of the crude lysates was used toinoculate a T80 flask with PER.C6 and virus was harvested at full CPE.The timeframe between inoculation and occurrence of CPE as well as theamount of virus needed to re-infect a new culture, differed betweenserotypes. Adenovirus stocks were prepared by freeze/thawing and used toinoculate 3-4 T175 cm² three-layer flasks with PER.C6 cells. Uponoccurrence of CPE, cells were harvested by tapping the flask, pelletedand virus was isolated and purified by a two-step CsCl gradient asfollows. Cell pellets were dissolved in 50 ml 10 mM NaPO₄ buffer (pH7.2) and frozen at −20° C. After thawing at 37° C., 5.6 ml sodiumdeoxycholate (5% w/v) was added. The solution was mixed gently andincubated for 5-15 minutes at 37° C. to completely lyse the cells. Afterhomogenizing the solution, 1875 μl 1M MgCl₂ was added. After theaddition of 375 μl DNAse (10 mg/ml) the solution was incubated for 30minutes at 37° C. Cell debris was removed by centrifugation at 1880×gfor 30 minutes at RT without brake. The supernatant was subsequentlypurified from proteins by extraction with FREON (3x). The clearedsupernatant was loaded on a 1M Tris/HCl buffered cesium chloride blockgradient (range: 1.2/1.4 g/ml) and centrifuged at 21000 rpm for 2.5hours at 10° C. The virus band is isolated after which a secondpurification using a 1 M Tris/HCl buffered continues gradient of 1.33g/ml of cesium chloride was performed. The virus was then centrifugedfor 17 hours at 55000 rpm at 10° C. The virus band is isolated andsucrose (50% w/v) is added to a final concentration of 1%. Excess cesiumchloride is removed by dialysis (three times 1 hr at RT) in dialysisslides (Slide-a-lizer, cut off 10000 kDa, Pierce, USA) against 1.5 literPBS supplemented with CaCl₂ (0.9 mM), MgCl₂ (0.5 mM) and an increasingconcentration of sucrose (1, 2, 5%). After dialysis, the virus isremoved from the slide-a-lizer after which it is aliquoted in portionsof 25 and 100 μl upon which the virus is stored at −85° C.

To determine the number of virus particles per milliliter, 50 μl of thevirus batch is run on a high-pressure liquid chromatograph (HPLC) asdescribed by Shabram et al (1997). Viruses were eluted using a NaClgradient ranging from 0 to 600 mM. As depicted in table I, the NaClconcentration by which the viruses were eluted differed significantlyamong serotypes.

Most human adenoviruses replicated well on PER.C6 cells with a fewexceptions. Adenovirus type 8 and 40 were grown on 911-E4 cells (He etal., 1998). Purified stocks contained between 5×10¹⁰ and 5×10¹² virusparticles/ml (VP/ml; see table I).

Titration of Purified Human Adenovirus Stocks

Adenoviruses were titrated on PER.C6 cells to determine the amount ofvirus necessary to obtain full CPE in five days, the length of theneutralization assay. Hereto, 100 μl medium was dispensed into each wellof 96-well plates. 25 μl of adenovirus stocks pre-diluted 10⁴, 10⁵, 10⁶or 10⁷ times were added to column 2 of a 96-well plate and mixed bypipetting up and down 10 times. Then 25 μl was brought from column 2 tocolumn 3 and again mixed. This was repeated until column 1 1 after which25 μl from column 11 was discarded. This way, serial dilutions in stepsof 5 were obtained starting off from a pre-diluted stock. Then 3×10⁴PER.C6 cells (ECACC deposit number 96022940) were added in a 100 μlvolume and the plates were incubated at 37° C., 5% CO₂ for five or sixdays. CPE was monitored microscopically. The method of Reed and Muenschwas used to calculate the cell culture-inhibiting dose 50% (CCID50).

In parallel, identical plates were set up that were analyzed using theMTT assay (Promega). In this assay, living cells are quantified bycalorimetric staining. Hereto, 20 μl MTT (7.5 mgr/ml in PBS) was addedto the wells and incubated at 37° C., 5% CO₂ for two hours. Thesupernatant was removed and 100 μl of a 20:1 isopropanol/triton-X100solution was added to the wells. The plates were put on a 96-well shakerfor 3-5 minutes to solubilize the precipitated staining. Absorbance wasmeasured at 540 nm and at 690 nm (background). By this assay, wells withproceeding CPE or full CPE can be distinguished.

Neutralization Assay

96-well plates with diluted human serum samples were thawed at 37° C.,5% CO₂. Adenovirus stocks diluted to 200 CCID50 per 50 μl were preparedand 50 μl aliquots were added to columns 1-11 of the plates with serum.Plates were incubated for 1 hour at 37° C., 5% CO₂. Then 50 μl PER.C6cells at 6×10⁵/ml were dispensed in all wells and incubated for 1 day at37° C., 5% CO2. Supernatant was removed using fresh pipette tips foreach row and 200 μl fresh medium was added to all wells to avoid toxiceffects of the serum. Plates were incubated for another 4 days at 37°C., 5% CO₂. In addition, parallel control plates were set up in duplowith diluted positive control sera generated in rabbits and specific foreach serotype to be tested in rows A and B and with negative controlserum (FCS) in rows C and D. Also, in each of the rows E-H a titrationwas performed as described above with steps of five times dilutionsstarting with 200 CCID50 of each virus to be tested. On day 5, one ofthe control plates was analyzed microscopically and with the MTT assay.The experimental titer was calculated from the control titration plateobserved microscopically. If CPE was found to be complete, i.e. thefirst dilution in the control titration experiment analyzed by MTT showsclear cell death, all assay plates were processed. If not, the assay wasallowed to proceed for one or more days until full CPE was apparentafter which all plates were processed. In most cases, the assay wasterminated at day 5. For Ad1, 5, 33, 39, 42 and 43 the assay was leftfor six days and for Ad2 for eight days.

A serum sample is regarded as “non-neutralizing” when, at the highestserum concentration, a maximum protection of 40% is seen compared tocontrols without serum.

The results of the analysis of 44 prototype adenoviruses against serumfrom 100 healthy volunteers are shown in FIG. 1. As expected, thepercentage of serum samples that contained neutralizing antibodies toAd2 and Ad5 was very high. This was also true for most of the lowernumbered adenoviruses. Surprisingly, none of the serum samples containedneutralizing antibodies to Ad35. Also, the number of individuals withneutralizing antibody titers to the serotypes 26, 34 and 48 was verylow. Therefore, recombinant E1-deleted adenoviruses based on Ad35 or oneof the other above mentioned serotypes have an important advantagecompared to recombinant vectors based on Ad5 with respect to clearanceof the viruses by neutralizing antibodies.

Also, Ad5-based vectors that have (parts of) the capsid proteinsinvolved in immunogenic response of the host replaced by thecorresponding (parts of) the capsid proteins of Ad35 or one of the otherserotypes will be less, or even not, neutralized by the vast majority ofhuman sera.

As can be seen in Table I, the VP/CCID50 ratio calculated from the virusparticles per ml and the CCID50 obtained for each virus in theexperiments was highly variable, and ranged from 0.4 to 5 log. This isprobably caused by different infection efficiencies of PER.C6 cells andby differences in replication efficiency of the viruses. Furthermore,differences in batch qualities may play a role. A high VP/CCID50 ratiomeans that more viruses were put in the wells to obtain CPE in 5 days.As a consequence, the outcome of the neutralization study might bebiased since more (inactive) virus particles could shield theantibodies. To check whether this phenomenon had taken place, theVP/CCID50 ratio was plotted against the percentage of serum samplesfound positive in the assay (FIG. 2). The graph clearly shows that thereis no negative correlation between the amount of viruses in the assayand neutralization in serum.

Example 2 The Prevalence of Neutralizing Activity (NA) to Ad35 is Low inHuman Sera from Different Geographic Locations

In Example 1 the analysis of neutralizing activity (“NA”) in human serafrom one location in Belgium was described. Strikingly, of a panel of 44adenovirus serotypes tested, one serotype, Ad35, was not neutralized inany of the 100 sera assayed. In addition, a few serotypes, Ad26, Ad34and Ad48 were found to be neutralized in 8%, or less, of the seratested. This analysis was further extended to other serotypes ofadenovirus not previously tested and, using a selection of serotypesfrom the first screen, was also extended to sera from differentgeographic locations.

Hereto, adenoviruses were propagated, purified and tested forneutralization in the CPE-inhibition assay as described in Example 1.Using the sera from the same batch as in Example 1, adenovirus serotypes7B, 11, 14, 18 and 44/1876 were tested for neutralization. These viruseswere found to be neutralized in, respectively, 59, 13, 30, 98 and 54% ofthe sera. Thus, of this series, Ad11 is neutralized with a relativelylow frequency.

Since it is known that the frequency of isolation of adenovirusserotypes from human tissue as well as the prevalence of NA toadenovirus serotypes may differ on different geographic locations, wefurther tested a selection of the adenovirus serotypes against sera fromdifferent places. Human sera were obtained from two additional places inEurope (Bristol, UK and Leiden, NL) and from two places in the UnitedStates (Stanford, Calif. and Great Neck, N.Y.). Adenoviruses that werefound to be neutralized in 20% or less of the sera in the first screen,as well as Ad2, Ad5, Ad27, Ad30, Ad38, Ad43, were tested forneutralization in sera from the UK. The results of these experiments arepresented in FIG. 3. Adenovirus serotypes 2 and 5 were again neutralizedin a high percentage of human sera. Furthermore, some of the serotypesthat were neutralized in a low percentage of sera in the first screenare neutralized in a higher percentage of sera from the UK, for example,Ad26 (7% vs. 30%), Ad28 (13% vs. 50%), Ad34 (5% vs. 27%) and Ad48 (8%vs. 32%). Neutralizing activity against Ad11 and Ad49 that were found ina relatively low percentage of sera in the first screen, are found in aneven lower percentage of sera in this second screen (13% vs. 5% and 20%vs. 11% respectively). Serotype Ad35 that was not neutralized in any ofthe sera in the first screen, was now found to be neutralized in a lowpercentage (8%) of sera from the UK. The prevalence of NA in human serafrom the UK is the lowest to serotypes Ad11 and Ad35.

For further analysis, sera obtained from two locations in the US(Stanford, Calif. and Great Neck, N.Y.) and from The Netherlands(Leiden). FIG. 4 presents an overview of data obtained with these seraand the previous data. Not all viruses were tested in all sera, exceptfor Ad5, Ad11 and Ad35. The overall conclusion from this comprehensivescreen of human sera is that the prevalence of neutralizing activity toAd35 is the lowest of all serotypes throughout the western countries: onaverage 7% of the human sera contain neutralizing activity (5 differentlocations). Another B-group adenovirus, Ad11 is also neutralized in alow percentage of human sera (average 11% in sera from 5 differentlocations). Adenovirus type 5 is neutralized in 56% of the human seraobtained from 5 different locations. Although not tested in all sera,D-group serotype 49 is also neutralized with relatively low frequency insamples from Europe and from one location of the US (average 14%).

In the herein described neutralization experiments, a serum is judgednon-neutralizing when, in the well with the highest serum concentration,the maximum protection of CPE is 40% compared to the controls withoutserum. The protection is calculated as follows:${\% \quad {protection}} = {\frac{{{OD}\quad {corresponding}\quad {well}} - {{OD}\quad {virus}\quad {control}}}{{{OD}\quad {non}\text{-}{infected}\quad {control}} - {{OD}\quad {virus}\quad {control}}} \times 100\%}$

As described in Example 1, the serum is plated in five differentdilutions ranging from 4× to 64× diluted. Therefore, it is possible todistinguish between low titers (i.e., neutralization only in the highestserum concentrations) and high titers of NA (i.e., also neutralizationin wells with the lowest serum concentration). Of the human sera used inour screen that were found to contain neutralizing activity to Ad5, 70%turned out to have high titers whereas of the sera that contained NA toAd35, only 15% had high titers. Of the sera that were positive for NA toAd11 only 8% had high titers. For Ad49, this was 5%. Therefore, not onlyis the frequency of NA to Ad35, Ad11 and Ad49 much lower as compared toAd5, but of the sera that do contain NA to these viruses, the vastmajority has low titers. Adenoviral vectors based on Ad 11, Ad35 or Ad49have therefore a clear advantage over Ad5 based vectors when used asgene therapy vehicles or vaccination vectors in vivo or in anyapplication where infection efficiency is hampered by neutralizingactivity.

In the following examples, the construction of a vector system for thegeneration of safe, RCA-free Ad35-based vectors is described.

Example 3 Sequence of the Human Adenovirus Type 35

Ad35 viruses were propagated on PER.C6 cells and DNA was isolated asfollows: To 100 μl of virus stock (Ad35: 3.26×10¹² VP/mil), 10μl 10×DNAse buffer (130 mM Tris-HCl pH7.5; 1,2 M CaCl₂; 50 mM MgCl₂) wasadded. After addition of 10 μl 10 mgr/ml DNAse I (Roche Diagnostics),the mixture was incubated for 1 hr. at 37° C. Following addition of 2.5μl 0.5M EDTA, 3.2 μl 20% SDS and 1.5μl ProteinaseK (Roche Diagnostics;20 mgr/ml), samples were incubated at 50° C. for 1 hr. Next, the viralDNA was isolated using the GENECLEAN spin kit (Bio101 Inc.) according tothe manufacturer's instructions. DNA was eluted from the spin columnwith 25 μl sterile MilliQ water. The total sequence was generated byQiagen Sequence Services (Qiagen GmbH, Germany). Total viral DNA wassheared by sonification and the ends of the DNA were made blunt by T4DNA polymerase. Sheared blunt fragments were size fractionated onagarose gels and gel slices corresponding to DNA fragments of 1.8 to 2.2kb were obtained. DNA was purified from the gel slices by the QIAquickgel extraction protocol and subcloned into a shotgun library of pUCl9plasmid cloning vectors. An array of clones in 96-well plates coveringthe target DNA 8 (+/−2) times was used to generate the total sequence.Sequencing was performed on Perkin-Elmer 9700 thermocyclers using BigDye Terminator chemistry and AmpliTaq FS DNA polymerase followed bypurification of sequencing reactions using QIAGEN DyeEx 96 technology.Sequencing reaction products were then subjected to automated separationand detection of fragments on ABI 377 XL 96 lane sequencers. Initialsequence results were used to generate a contiguous sequence and gapswere filled in by primer walking reads on the target DNA or by directsequencing of PCR products. The ends of the virus turned out to beabsent in the shotgun library, most probably due to cloning difficultiesresulting from the amino acids of pTP that remain bound to the ITRsequences after proteinase K digestion of the viral DNA. Additionalsequence runs on viral DNA solved most of the sequence in those regions,however it was difficult to obtain a clear sequence of the most terminalnucleotides. At the 5′ end the sequence portion obtained was5′-CCAATAATATACCT-3′ (SEQ ID NO: 1) while at the 3′ end, the obtainedsequence portion was 5′-AGGTATATTATTGATGATGGG-3′ (SEQ ID NO: 2). Mosthuman adenoviruses have a terminal sequence 5′-CATCATCAATAATATACC-3′(SEQ ID NO: 3). In addition, a clone representing the 3′ end of the Ad35DNA obtained after cloning the terminal 7 kb Ad35 EcoRI fragment intopBr322 also turned out to have the typical CATCATCAATAAT . . . sequence.Therefore, Ad35 may have the typical end sequence and the differencesobtained in sequencing directly on the viral DNA are due to artefactscorrelated with run-off sequence runs and the presence of residual aminoacids of pTP.

The total sequence of Ad35 with corrected terminal sequences is given inFIG. 5 and identified as SEQ ID NO: 39 in the SEQUENCE LISTING. Basedsequence homology with Ad5 (Genbank #M72360) and Ad7 (partial sequenceGenbank #X03000) and on the location of open reading frames, theorganization of the virus is identical to the general organization ofmost human adenoviruses, especially the subgroup B viruses. The totallength of the genome is 34,794 basepairs.

Example 4 Construction of a Plasmid-based Vector System to GenerateRecombinant Ad35-based Viruses

A functional plasmid-based vector system to generate recombinantadenoviral vectors comprises the following components:

1. An adapter plasmid comprising a left ITR and packaging sequencesderived from Ad35 and at least one restriction site for insertion of anheterologous expression cassette and lacking E1 sequences. Furthermore,the adapter plasmid contains Ad35 sequences 3′ from the E1B codingregion including the pIX promoter and coding sequences enough to mediatehomologous recombination of the adapter plasmid with a second nucleicacid molecule.

2. A second nucleic acid molecule, comprising sequences homologous tothe adapter plasmid, and Ad35 sequences necessary for the replicationand packaging of the recombinant virus, that is early, intermediate andlate genes that are not present in the packaging cell.

3. A packaging cell providing at least functional E1 proteins capable ofcomplementing the E1 function of Ad35.

Other methods for generating recombinant adenoviruses on complementingpackaging cells are known in the art, and may be applied to Ad35 viruseswithout departing from the invention. As an example, the construction ofa plasmid-based system, as outlined above, is described in detail below.

1) Construction of Ad35 Adapter Plasmids.

The adapter plasmid pAdApt (described in International PatentApplication WO99/55 132) was first modified to obtain adapter plasmidsthat contain extended polylinkers and that have convenient uniquerestriction sites flanking the left ITR and the adenovirus sequence atthe 3′ end to enable liberation of the adenovirus insert from plasmidvector sequences. Construction of these plasmids is described below indetail:

Adapter plasmid pAdApt was digested with SalI and treated with ShrimpAlkaline Phosphatase to reduce religation. A linker, composed of thefollowing two phosphorylated and annealed oligos: ExSalPacF 5′-TCG ATGGCA AAC AGC TAT TAT GGG TAT TAT GGG TTC GAA TTA ATT AA-3′ (SEQ ID NO: 4)and ExSalPacR 5′-TCG ATT AAT TAA TTC GAA CCC ATA ATA CCC ATA ATA GCT GTTTGC CA-3′ (SEQ ID NO: 5) was directly ligated into the digestedconstruct, thereby replacing the SalI restriction site by Pi-PspI, SwaIand PacI. This construct was designated pADAPT+ExSalPac linker.Furthermore, part of the left ITR of pAdApt was amplified by PCR usingthe following primers: PCLIPMSF: 5′-CCC CAA TTG GTC GAC CAT CAT CAA TAATAT ACC TTA TTT TGG-3′ (SEQ ID NO: 6) and pCLIPBSRGI: 5′-GCG AAA ATT GTCACT TCC TGT G-3′ (SEQ ID NO: 7). The amplified fragment was digestedwith MunI and BsrGI and cloned into pAd5/Clip (described inInternational Patent Application WO99/55132), which was partiallydigested with EcoRI and after purification digested with BsrGI, therebyre-inserting the left ITR and packaging signal. After restriction enzymeanalysis, the construct was digested with ScaI and SgrAI and an 800 bpfragment was isolated from gel and ligated into ScaI/SgrAI digestedpADAPT+ExSalPac linker. The resulting construct, designatedpIPspSalAdapt, was digested with SalI, dephosphorylated, and ligated tothe phosphorylated ExSalPacF/ExSalPacR double-stranded linker previouslymentioned. A clone in which the PacI site was closest to the ITR wasidentified by restriction analysis and sequences were confirmed bysequence analysis. This novel pAdApt construct, termed pIPspAdapt thusharbours two ExSalPac linkers containing recognition sequences for PacI,PI-PspI and BstBI, which surround the adenoviral part of the adenoviraladapter construct, and which can be used to linearize the plasmid DNAprior to cotransfection with adenoviral helper fragments.

In order to further increase transgene cloning permutations, a number ofpolylinker variants were constructed based on pIPspAdapt. For thispurpose, pIPspAdapt was first digested with EcoRI and dephosphorylated.A linker composed of the following two phosphorylated and annealedoligos: Ecolinker+: 5′-AAT TCG GCG CGC CGT CGA CGA TAT CGA TAG CGG CCGC-3′(SEQ ID NO: 8) and Ecolinker−: 5′-AAT TGC GGC CGC TAT CGA TAT CGTCGA CGG CGC GCC G-3′ (SEQ ID NO: 9) was ligated into this construct,thereby creating restriction sites for AscI, SalI, EcoRV, ClaI and NotI.Both orientations of this linker were obtained, and sequences wereconfirmed by restriction analysis and sequence analysis. The plasmidcontaining the polylinker in the order 5′ HindIII, KpnI, Agel, EcoRI,AscI, SalI, EcoRV, ClaI, NotI, NheI, HpaI, BamHI and XbaI was termedpIPspAdapt1 while the plasmid containing the polylinker in the orderHindIII, KpnI, AgeI, NotI, ClaI, EcoRV, SalI, AscI, EcoRI, NheI, HpaI,BamHI and XbaI was termed pIPspAdapt2.

To facilitate the cloning of other sense or antisense constructs, alinker composed of the following two oligonucleotides was designed, toreverse the polylinker of pIPspAdapt: HindXba+5′-AGC TCT AGA GGA TCC GTTAAC GCT AGC GAA TTC ACC GGT ACC AAG CTT A-3′ (SEQ ID NO: 10);HindXba-5′-CTA GTA AGC TTG GTA CCG GTG AAT TCG CTA GCG TTA ACG GAT CCTCTA G-3′ (SEQ ID NO: 11). This linker was ligated into HindIII/XbaIdigested pIPspAdapt and the correct construct was isolated. Confirmationwas done by restriction enzyme analysis and sequencing. This newconstruct, pIPspAdaptA, was digested with EcoRI and the previouslymentioned Ecolinker was ligated into this construct. Both orientationsof this linker were obtained, resulting in pIPspAdapt3, which containsthe polylinker in the order XbaI, BamHI, HpaI, NheI, EcoRI, AscI, SalI,EcoRV, ClaI, NotI, Agel, KpnI and HindIII. All sequences were confirmedby restriction enzyme analysis and sequencing.

Adapter plasmids based on Ad35 were then constructed as follows:

The left ITR and packaging sequence corresponding to Ad35 wt sequencesnucleotides 1 to 464 (FIG. 5) were amplified by PCR on wtAd35 DNA usingthe following primers:

Primer 35F1:

5′-CGG AAT TCT TAA TTA ATC GAC ATC ATC AAT AAT ATA CCT TAT AG-3′ (SEQ IDNO: 12)

Primer 35R2:

5′-GGT GGT CCT AGG CTG ACA CCT ACG TAA AAA CAG-3′ (SEQ ID NO: 13)

Amplification introduces a PacI site at the 5′ end and an AvrII site atthe 3′ end of the sequence.

For the amplification, Platinum Pfx DNA polymerase enzyme (LTI) was usedaccording to manufacturer's instructions, but with primers at 0.6 μM andwith DMSO added to a final concentration of 3%. Amplification programwas as follows: 2 min. at 94° C., (30 sec. 94° C., 30 sec. at 56° C., 1min. at 68° C.) for 30 cycles, followed by 10 min. at 68° C.

The PCR product was purified using a PCR purification kit (LTI)according to the manufacturer's instructions, and digested with PacI andAvrII. The digested fragment was then purified from gel using theGENECLEAN kit (Bio 101, Inc.). The Ad5-based adapter plasmidpIPspAdApt-3 was digested with AvrII and then partially with PacI andthe 5762 bp fragment was isolated in an LMP agarose gel slice andligated with the abovementioned PCR fragment digested with the sameenzymes and transformed into electrocompetent DH10B cells (LTI). Theresulting clone is designated pIPspAdApt3-Ad351ITR.

In parallel, a second piece of Ad35 DNA was amplified using thefollowing primers:

35F3: 5′-TGG TGG AGA TCT GGT GAG TAT TGG GAA AAC-3′ (SEQ ID NO: 14)

35R4: 5′-CGG AAT TCT TAA TTA AGG GAA ATG CAA ATC TGT GAG G-3′ (SEQ IDNO: 15)

The sequence of this fragment corresponds to nucleotides 3401 to 4669 ofwtAd35 (FIG. 5) and contains 1.3 kb of sequences starting directly 3′from the E1B 55k coding sequence. Amplification and purification weredone as previously described herein for the fragment containing the leftITR and packaging sequence. The PCR fragment was then digested with PacIand subcloned into pNEB 193 vector (New England Biolabs) digested withSmal and PacI. The integrity of the sequence of the resulting clone waschecked by sequence analysis. pNEB/Ad35pF3R4 was then digested withBglII and PacI and the Ad35 insert was isolated from gel using theQIAExII kit (Qiagen). pIPspAdApt3-Ad351ITR was digested with BglII andthen partially with PacI. The 3624 bp fragment (containing vectorsequences, the Ad35 ITR and packaging sequences as well as the CMVpromoter, multiple cloning region and polyA signal) was also isolatedusing the QIAExII kit (Qiagen). Both fragments were ligated andtransformed into competent DH10B cells (LTI). The resulting clone,pAdApt35IP3, has the expression cassette from pIPspAdApt3 but containsthe Ad35 left ITR and packaging sequences and a second fragmentcorresponding to nucleotides 3401 to 4669 from Ad35. A second version ofthe Ad35 adapter plasmid having the multiple cloning site in theopposite orientation was made as follows:

pIPspAdapt1 was digested with NdeI and BglII and the 0.7 kbp bandcontaining part of the CMV promoter, the MCS and SV40 polyA was isolatedand inserted in the corresponding sites of pAdApt35IP3 generatingpAdApt35IP1 (FIG. 6).

pAdApt35.LacZ and pAdApt35.Luc adapter plasmids were then generated byinserting the transgenes from pcDNA.LacZ (digested with KpnI and BamHI)and pAdApt.Luc (digested with HindIII and BamHI) into the correspondingsites in pAdApt35IP1. The generation of pcDNA.LacZ and pAdApt.Luc isdescribed in International Patent Application WO99/55132.

2) Construction of Cosmid pWE.Ad35.pIX-rITR

FIG. 7 presents the various steps undertaken to construct the cosmidclone containing Ad35 sequences from bp 3401 to 34794 (end of the rightITR) that are described in detail below.

A first PCR fragment (pIX-NdeI) was generated using the following primerset:

35F5: 5′-CGG AAT TCG CGG CCG CGG TGA GTA TTG GGA AAA C-3′ (SEQ ID NO:16)

35R6: 5′-COC CAG ATC GTC TAC AGA ACA G-3′ (SEQ ID NO: 17)

DNA polymerase Pwo (Roche) was used according to manufacturer'sinstructions, however, with an end concentration of 0.6 pM of bothprimers and using 50 ngr wt Ad35 DNA as template. Amplification was doneas follows: 2 min. at 94° C., 30 cycles of 30 sec. at 94° C., 30 sec. at65° C. and 1 min. 45 sec. at 72° C., follow at 68° C. To enable cloningin the TA cloning vector PCR2.1, a last incubation with 1 unit superTaqpolymerase (HT Biotechnology LTD) for 10 min. at 72° C. was performed.

The 3370 bp amplified fragment contains Ad35 sequences from bp 3401 to6772 with a NotI site added to the 5′ end. Fragments were purified usingthe PCR purification kit (LTI).

A second PCR fragment (NdeI-rITR) was generated using the followingprimers:

35F7: 5′-GAA TGC TGG CTT CAG TTG TAA TC-3′ (SEQ ID NO: 18)

35R8: 5′-CGG AAT TCG CGG CCG CAT TTA AAT CAT CAT CAA TAA TAT ACC-3′ (SEQID NO: 19)

Amplification was done with pfx DNA polymerase (LTI) according tomanufacturer's instructions but with 0.6 lM of both primers and 3% DMSOusing 10 ngr. of wtAd35 DNA as template. The program was as follows: 3min. at 94° C. and 5 cycles of 30 sec. at 94° C., 45 sec. at 40° C., 2min.45 sec. at 68° C. followed by 25 cycles of 30 sec. at 94° C., 30sec. at 60° C., 2 min.45 sec. at 68° C. To enable cloning in theTA-cloning vector PCR2.1, a last incubation with 1 unit superTaqpolymerase for 10 min. at 72° C. was performed. The 1.6 kb amplifiedfragment ranging from nucleotides 33178 to the end of the right ITR ofAd35, was purified using the PCR purification kit (LTI).

Both purified PCR fragments were ligated into the PCR2.1 vector of theTA-cloning kit (Invitrogen) and transformed into STBL-2 competent cells(LTD). Clones containing the expected insert were sequenced to confirmcorrect amplification. Next, both fragments were excised from the vectorby digestion with NotI and NdeI and purified from gel using theGENECLEAN kit (BIO 101, Inc.). Cosmid vector pWE15 (Clontech) wasdigested with NotI, dephosphorylated and also purified from gel. Thesethree fragments were ligated and transformed into STBL2 competent cells(LTI). One of the correct clones that contained both PCR fragments wasthen digested with NdeI, and the linear fragment was purified from gelusing the GENECLEAN kit. Ad35 wt DNA was digested with NdeI and the 26.6kb fragment was purified from LMP gel using agarase enzyme (Roche)according to the manufacturer's instructions. These fragments wereligated together and packaged using λl phage packaging extracts(Stratagene) according to the manufacturer's protocol. After infectioninto STBL-2 cells, colonies were grown on plates and analysed forpresence of the complete insert. One clone with the large fragmentinserted in the correct orientation and having the correct restrictionpatterns after independent digestions with three enzymes (NcoI, PvuIIand ScaI) was selected. This clone is designated pWE.Ad35.pIX-rITR. Itcontains the Ad35 sequences from bp 3401 to the end and is flanked byNotI sites (FIG. 8).

3) Generation of Ad35 Based Recombinant Viruses on PER.C6.

Wild type Ad35 virus can be grown on PER.C6 packaging cells to very hightiters. However, whether the Ad5-E1 region that is present in PER.C6 isable to complement E1-deleted Ad35 recombinant viruses is unknown. Totest this, PER.C6 cells were cotransfected with the above describedadapter plasmid pAdApt35.LacZ and the large backbone fragmentpWE.Ad35.pIX-rITR. First, pAdApt35.LacZ was digested with PacI andpWE.Ad35.pIX-rITR was digested with NotI. Without further purification,4 μgr of each construct was mixed with DMEM (LTI) and transfected intoPER.C6 cells, seeded at a density of 5×10⁶ cells in a T25 flask the daybefore, using Lipofectamin (LTI) according to the manufacturer'sinstructions. As a positive control, 6μgr of PacI digestedpWE.Ad35.pIX-rITR DNA was cotransfected with a 6.7 kb NheI fragmentisolated from Ad35 wt DNA containing the left end of the viral genomeincluding the E1 region. The next day, medium (DMEM with 10% FBS and 10mM MgCl₂) was refreshed and cells were further incubated. At day 2following the transfection, cells were trypsinized and transferred toT80 flasks. The positive control flask showed CPE at five days followingtransfection, showing that the pWE.Ad35.pIX-rITR construct is functionalat least in the presence of Ad35-E1 proteins. The transfection with theAd35 LacZ adapter plasmid and pWE.Ad35.pIX-rITR did not give rise toCPE. These cells were harvested in the medium at day 10 andfreeze/thawed once to release virus from the cells. 4 ml of theharvested material was added to a T80 flask with PER.C6 cells (at 80%confluency) and incubated for another five days. Thisharvest/re-infection was repeated for two times but there was noevidence for virus associated CPE.

From this experiment, it seems that the Ad5-E1 proteins are not, or notwell enough, capable of complementing Ad35 recombinant viruses, however,it may be that the sequence overlap of the adapter plasmid and thepWE.Ad35.pIX-rITR backbone plasmid is not large enough to efficientlyrecombine and give rise to a recombinant virus genome. The positivecontrol transfection was done with a 6.7 kb left end fragment andtherefore the sequence overlap was about 3.5 kb. The adapter plasmid andthe pWE.Ad35.pIX-rITR fragment have a sequence overlap of 1.3 kb. Tocheck whether the sequence overlap of 1.3 kb is too small for efficienthomologous recombination, a cotransfection was done with PacI digestedpWE.Ad35.pIX-rITR and a PCR fragment of Ad35 wt DNA generated with theabove mentioned 35F1 and 35R4 using the same procedures as previouslydescribed herein. The PCR fragment thus contains left end sequences upto bp 4669 and, therefore, has the same overlap sequences withpWE.Ad35.pIX-rITR as the adapter plasmid pAdApt35.LacZ, but has Ad35 E1sequences. Following PCR column purification, the DNA was digested withSalI to remove possible intact template sequences. A transfection withthe digested PCR product alone served as a negative control. Four daysafter the transfection, CPE occurred in the cells transfected with thePCR product and the Ad35 pIX-rITR fragment, and not in the negativecontrol. This result shows that a 1.3 kb overlapping sequence issufficient to generate viruses in the presence of Ad35 E1 proteins. Fromthese experiments, we conclude that the presence of at least one of theAd3 5.E1 proteins is necessary to generate recombinant Ad35 basedvectors from plasmid DNA on Ad5 complementing cell lines.

Example 5

1) Construction of Ad35.E1 Expression Plasmids

Since Ad5-E1 proteins in PER.C6 are incapable of complementing Ad35recombinant viruses efficiently, Ad35 E1 proteins have to be expressedin Ad5 complementing cells (e.g., PER.C6). Alternatively, a newpackaging cell line expressing Ad35 E1 proteins has to be made, startingfrom either diploid primary human cells or established cell lines notexpressing adenovirus E1 proteins. To address the first possibility, theAd35 E1 region was cloned in expression plasmids as described below.

First, the Ad35 E1 region from bp 468 to bp 3400 was amplified fromwtAd35 DNA using the following primer set:

35F11: 5′-GGG GTA CCG AAT TCT CGC TAG GGT ATT TAT ACC-3′ (SEQ ID NO: 20)

35F10: 5′-GCT CTA GAC CTG CAG GTT AGT CAG TTT CTT CTC CAC TG-3′ (SEQ IDNO: 21)

This PCR introduces a KpnI and EcoRI site at the 5′ end and an SbfI andXbaI site at the 3′ end.

Amplification on 5 ngr. template DNA was done with Pwo DNA polymerase(Roche) using the manufacturer's instructions, however, with bothprimers at a final concentration of 0.6 μM. The program was as follows:2 min. at 94° C., 5 cycles of 30 sec. at 94° C., 30 sec. at 56° C. and 2min. at 72° C., followed by 25 cycles of 94° C., 30 sec. at 60° C. and 2min. at 72° C., followed by 10 min. at 72° C. PCR product was purifiedby a PCR purification kit (LTI) and digested with KpnI and XbaI. Thedigested PCR fragment was then ligated to the expression vectorpRSVhbvNeo (see below) also digested with KpnI and XbaI. Ligations weretransformed into competent STBL-2 cells (LTI) according tomanufacturer's instructions and colonies were analysed for the correctinsertion of Ad35E1 sequences into the polylinker in between the RSVpromoter and HBV polyA.

The resulting clone was designated pRSV.Ad35-E1 (FIG. 9). The Ad35sequences in pRSV.Ad35-E1 were checked by sequence analysis.

pRSVhbvNeo was generated as follows: pRc-RSV (Invitrogen) was digestedwith PvuII, dephosphorylated with TSAP enzyme (LTI), and the 3 kb vectorfragment was isolated in low melting point agarose (LMP). PlasmidpPGKneopA (FIG. 10; described in International Patent ApplicationWO96/35798) was digested with SspI completely to linearize the plasmidand facilitate partial digestion with PvuII. Following the partialdigestion with PvuII, the resulting fragments were separated on a LMPagarose gel and the 2245 bp PvuII fragment, containing the PGK promoter,neomycin-resistance gene and HBVpolyA, was isolated. Both isolatedfragments were ligated to give the expression vector pRSV-pNeo that nowhas the original SV40prom-neo-SV40polyA expression cassette replaced bya PGKprom-neo-HBVpolyA cassette (FIG. 11). This plasmid was furthermodified to replace the BGHpA with the HBVpA as follows: pRSVpNeo waslinearised with ScaI and further digested with XbaI. The 1145 bpfragment, containing part of the Amp gene and the RSV promoter sequencesand polylinker sequence, was isolated from gel using the GeneClean kit(Bio Inc. 101). Next, pRSVpNeo was linearised with ScaI and furtherdigested with EcoRI partially and the 3704 bp fragment containing thePGKneo cassette and the vector sequences were isolated from gel asabove. A third fragment, containing the HBV polyA sequence flanked byXbaI and EcoRI at the 5′ and 3′ end respectively, was then generated byPCR amplification on pRSVpNeo using the following primer set:

HBV-F: 5′-GGC TCT AGA GAT CCT TCG CGG GAC GTC-3′ (SEQ ID NO: 22) and

HBV-R: 5′-GGC GAA TTC ACT GCC TTC CAC CAA GC-3′ (SEQ ID NO: 23).

Amplification was done with Elongase enzyme (LTI) according to themanufacturer's instructions with the following conditions: 30 seconds at94° C., then 5 cycles of 45 seconds at 94° C., 1 minute at 42° C. and 1minute 68° C., followed by 30 cycles of 45 seconds at 94° C., 1 minuteat 65° C. and 1 minute at 68° C., followed by 10 minutes at 68° C. The625 bp PCR fragment was then purified using the Qiaquick PCRpurification kit, digested with EcoRI and XbaI and purified from gelusing the GENECLEAN kit. The three isolated fragments were ligated andtransformed into DH5α competent cells (LTI) to give the constructpRSVhbvNeo (FIG. 12). In this construct, the transcription regulatoryregions of the RSV expression cassette and the neomycin selection markerare modified to reduce overlap with adenoviral vectors that oftencontain CMV and SV40 transcription regulatory sequences.

2) Generation of Ad35 Recombinant Viruses on PER.C6 Cells Cotransfectedwith an Ad35-E1 Expression Construct.

PER.C6 cells were seeded at a density of 5×10⁶ cells in a T25 flask and,the next day, transfected with a DNA mixture containing:

1 μg pAdApt35.LacZ digested with PacI

1 μg pAdApt35.LacZ digested with PacI

5 μg pRSV.Ad35E1 undigested

2 μg pWE.Ad35.pIX-aITR digested with NotI

Transfection was done using Lipofectamine according to themanufacturer's instructions. Five hours after addition of thetransfection mixture to the cells, medium was removed and replaced byfresh medium. After two days, cells were transferred to T80 flasks andfurther cultured. One week post-transfection, 1 ml of the medium wasadded to A549 cells and, the following day, cells were stained for LacZexpression. Blue cells were clearly visible after two hours of stainingindicating that recombinant LacZ expressing viruses were produced. Thecells were further cultured, but no clear appearance of CPE was noted.However, after 12 days, clumps of cells appeared in the monolayer and 18days following transfection, cells were detached. Cells and medium werethen harvested, freeze-thawed once, and 1 ml of the crude lysate wasused to infect PER.C6 cells in a 6-well plate. Two days after infection,cells were stained for LacZ activity. After two hours, 15% of the cellswere stained blue. To test for the presence of wt and/or replicatingcompetent viruses, A549 cells were infected with these virused andfurther cultures. No signs of CPE were stained blue. To test for thereplication competent viruses. These experiments show that recombinantAdApt35.LacZ viruses were made on PER.C6 cells cotransfected with anAd35-E1 expression construct.

Ad35 recombinant viruses escape neutralization in human serum containingneutralizing activity to Ad5 viruses.

The AdApt35.LacZ viruses were then used to investigate infection in thepresence of serum that contains neutralizing activity to Ad5 viruses.Purified Ad5-based LacZ virus served as a positive control for NA.Hereto, PER.C6 cells were seeded in a wells plate at a density of 2×10⁵cells/well. The next day, a human serum sample with high neutralizingactivity to Ad5 was diluted in culture medium in five steps of fivetimes dilutions. 0.5 ml of diluted serum was then mixed with 4×10⁶ virusparticles AdApt5.LacZ virus in 0.5 ml medium and after 30 minutes ofincubation at 37° C., 0.5 ml of the mixture was added to PER.C6 cells induplicate. For the AdApt35.LacZ viruses, 0.5 ml of the diluted serumsamples were mixed with 0.5 ml crude lysate containing AdApt35.LacZvirus and after incubation 0.5 ml of this mixture was added to PER.C6cells in duplo. Virus samples incubated in medium without serum wereused as positive controls for infection. After two hours of infection at37° C., medium was added to reach a final volume of 1 ml and cells werefurther incubated. Two days after infection, cells were stained for LacZactivity. The results are shown in Table II. From these results, it isclear that whereas AdApt5.LasZ viruses are efficiently neutralized,AdApt35.LasZ viruses remain infectious irrespective of the presence ofhuman serum. This proves that recombinant Ad3 5-based viruses escapeneutralization in human sera that contain NA to Ad5-based viruses.

Example 6 Generation of Cell Lines Capable of Complementing E1-deletedAd35 Viruses Generation of pIG135 and pIG270

Construct pIG.E1A.E1B (FIG. 13) contains E1 region sequences of Ad5corresponding to nucleotides 459 to 3510 of the wt Ad5 sequence (Genbankaccession number M72360) operatively linked to the humanphosphoglycerate kinase promoter (“PGK”) and the Hepatitis B Virus polyAsequences. The generation of this construct is described inInternational Patent Application No. WO97/00326. The E1 sequences of Ad5were replaced by corresponding sequences of Ad35 as follows.pRSV.Ad35-E1 (described in Example 5) was digested with EcoRI andSse8387I and the 3 kb fragment corresponding to the Ad35 E1 sequenceswas isolated from gel. Construct pIG.E1A.E1B was digested with Sse8387Icompletely and partially with EcoRI. The 4.2 kb fragment correspondingto vector sequences without the Ad5 E1 region but retaining the PGKpromoter were separated from other fragments on LMP agarose gel and thecorrect band was excised from gel. Both obtained fragments were ligatedresulting in pIG.Ad35-E1.

This vector was further modified to remove the LacZ sequences present inthe pUC119 vector backbone. Hereto, the vector was digested with BsaAIand BstXI and the large fragment was isolated from gel. A doublestranded oligo was prepared by annealing the following two oligos:

BB1: 5′-GTG CCT AGG CCA CGG GG-3′ (SEQ ID NO: 24) and

BB2: 5′-GTG GCC TAG GCA C-3′ (SEQ ID NO: 25).

Ligation of the oligo and the vector fragment resulted in constructpIG135 (FIG. 14). Correct insertion of the oligo restores the BsaAI andBstXI sites and introduces a unique AvrII site. Next, we introduced aunique site at the 3′ end of the Ad35-E1 expression cassette in pIG135.Hereto, the construct was digested with Sapi and the 3′ protruding endswere made blunt by treatment with T4 DNA polymerase. The thus treatedlinear plasmid was further digested with BsrGI and the largevector-containing fragment was isolated from gel. To restore the 3′ endof the HBVpolyA sequence and to introduce a unique site, a PCR fragmentwas generated using the following primers:

270F: 5′-CAC CTC TGC CTA ATC ATC TC-3′ (SEQ ID NO: 26) and

270R: 5′-GCT CTA GAA ATT CCA CTG CCT TCC ACC-3′ (SEQ ID NO: 27).

The PCR was performed on pIG.Ad35.E1 DNA using Pwo polymerase (Roche)according to the manufacturer's instructions. The obtained PCR productwas digested with BsrGI and dephosphorylated using Tsap enzyme (LTI),the latter to prevent insert dimerization on the BsrGI site. The PCRfragment and the vector fragment were ligated to yield construct pIG270(FIG. 15).

Ad35 E1 Sequences are Capable of Transforming Rat Primary Cells

New born WAG/RIJ rats were sacrificed at 1 week of gestation and kidneyswere isolated. After careful removal of the capsule, kidneys weredisintegrated into a single cell suspension by multiple rounds ofincubation in trypsin/EDTA (LTI) at 37° C. and collection of floatingcells in cold PBS containing 1% FBS. When most of the kidney wastrypsinized all cells were re-suspended in DMEM supplemented with 10%FBS and filtered through a sterile cheesecloth. Baby Rat Kidney (BRK)cells obtained from one kidney were plated in 5 dishes (Greiner, 6 cm).When a confluency of 70-80% was reached, the cells were transfected with1 or 5 μgr DNA/dish using the CaPO₄ precipitation kit (LTI) according tothe manufacturer's instructions. The following constructs were used inseparate transfections: pIG.E1A.E1B (expressing the Ad5-E1 region),pRSV.Ad35-E1, pIG.Ad35-E1 and pIG270 (expressing the Ad35-E1 region).Cells were incubated at 37° C., 5% CO₂ until foci of transformed cellsappeared. Table III shows the number of foci that resulted from severaltransfection experiments using circular or linear DNA. As expected, theAd5-E1 region efficiently transformed BRK cells. Foci also appeared inthe Ad35-E1 transfected cell layer although with lower efficiency. TheAd35 transformed foci appeared at a later time point: ˜2 weeks posttransfection compared with 7-10 days for Ad5-E1. These experimentsclearly show that the E1 genes of the B group virus Ad35 are capable oftransforming primary rodent cells. This proves the functionality of theAd35-E1 expression constructs and confirms earlier findings of thetransforming capacity of the B-group viruses Ad3 and Ad7 (Dijkema,1979). To test whether the cells in the foci were really transformed afew foci were picked and expanded. From the 7 picked foci at least 5turned out to grow as established cell lines.

Generation of New Packaging Cells Derivedfrom Primary Human Amniocytes

Amniotic fluid obtained after amniocentesis was centrifuged and cellswere re-suspended in AmnioMax medium (LTI) and cultured in tissueculture flasks at 37° C. and 10% CO₂. When cells were growing nicely(approximately one cell division/24 hrs.), the medium was replaced witha 1:1 mixture of AmnioMax complete medium and DMEM low glucose medium(LTI) supplemented with Glutamax I (end concentration 4 mM, LTI) andglucose (end concentration 4.5 gr/L, LTI) and 10% FBS (LTI). Fortransfection ˜5×10⁵ cells were plated in 10 cm tissue culture dishes.The day after, cells were transfected with 20 lgr of circularpIG270/dish using the CaPO₄ transfection kit (LTI) according tomanufacturer's instructions and cells were incubated overnight with theDNA precipitate. The following day, cells were washed 4 times with PBSto remove the precipitate and further incubated for over three weeksuntil foci of transformed cells appeared. Once a week the medium wasreplaced by fresh medium. Other transfection agents like, but notlimited to, LipofectAmine (LTI) or PEI (Polyethylenimine, high molecularweight, water-free, Aldrich) were used. Of these three agents PEIreached the best transfection efficiency on primary human amniocytes:˜1% blue cells 48 hrs. Following transfection of pAdApt35. LacZ.

Foci are isolated as follows. The medium is removed and replaced by PBSafter which foci are isolated by gently scraping the cells using a50-200 μl Gilson pipette with a disposable filter tip. Cells containedin ˜10 μl PBS were brought in a 96 well plate containing 15 μltrypsin/EDTA (LTI) and a single cell suspension was obtained bypipetting up and down and a short incubation at room temperature. Afteraddition of 200 μl of the above described 1:1 mixture of AmnioMaxcomplete medium and DMEM with supplements and 10% FBS, cells werefurther incubated. Clones that continued to grow were expanded andanalysed their ability to complement growth of E1-deleted adenoviralvectors of different sub-groups, specifically ones derived from B-groupviruses specifically from Ad35 or Ad11.

Generation of New Packaging Cell Lines from HER Cells

HER cells are isolated and cultured in DMEM medium supplemented with 10%FBS (LTI). The day before transfection, ˜5×10⁵ cells are plated in 6 cmdishes and cultured overnight at 37° C. and 10% CO₂. Transfection isdone using the CaPO₄ precipitation kit (LTI) according to themanufacturer's instructions. Each dish is transfected with 8-10 μmgrpIG270DNA, either as a circular plasmid or as a purified fragment. Toobtain the purified fragment, pIG270 was digested with AvrII and XbaIand the 4 kb fragment corresponding to the Ad35 E1 expression cassettewas isolated from gel by agarase treatment (Roche). The following day,the precipitate is washed away carefully by four washes with sterilePBS. Then fresh medium is added and transfected cells are furthercultured until foci of transformed cells appear. When large enough (>100cells) foci are picked and brought into 96-wells as described above.Clones of transformed HER cells that continue to grow, are expanded andtested for their ability to complement growth of E1-deleted adenoviralvectors of different sub-groups specifically ones derived from B-groupviruses specifically from Ad35 or Ad11.

New Packaging Cell Lines Derivedfrom PER.C6

As described in Example 5, it is possible to generate and grow Ad35E1-deleted viruses on PER.C6 cells with cotransfection of an Ad35-E1expression construct, e.g. pRSV.Ad35.E1. However, large-scale productionof recombinant adenoviruses using this method is cumbersome because, foreach amplification step, a transfection of the Ad35-E1 construct isneeded. In addition, this method increases the risk of non-homologousrecombination between the plasmid and the virus genome with high chancesof generation of recombinant viruses that incorporate E1 sequencesresulting in replication competent viruses. To avoid this, theexpression of Ad35-E1 proteins in PER.C6 has to be mediated byintegrated copies of the expression plasmid in the genome. Since PER.C6cells are already transformed and express Ad5-E1 proteins, addition ofextra Ad35-E1 expression may be toxic for the cells, however, it is notimpossible to stably transfect transformed cells with E1 proteins sinceAd5-E1 expressing A549 cells have been generated.

In an attempt to generate recombinant adenoviruses derived from subgroupB virus Ad7, Abrahamsen et al. (1997) were not able to generateE1-deleted viruses on 293 cells without contamination of wt Ad7. Virusesthat were picked after plaque purification on 293-ORF6 cells (Brough etal., 1996) were shown to have incorporated Ad7 E1B sequences bynon-homologous recombination. Thus, efficient propagation of Ad7recombinant viruses proved possible only in the presence of Ad7-E1Bexpression and Ad5-E4-ORF6 expression. The E1B proteins are known tointeract with cellular as well as viral proteins (Bridge et al., 1993;White, 1995). Possibly, the complex formed between the E1B 55K proteinand E4-ORF6 which is necessary to increase mRNA export of viral proteinsand to inhibit export of most cellular mRNAs, is critical and in someway serotype specific. The above experiments suggest that the E1Aproteins of Ad5 are capable of complementing an Ad7-E1A deletion andthat Ad7-E1B expression in adenovirus packaging cells on itself is notenough to generate a stable complementing cell line. To test whether oneor both of the Ad35-E1B proteins is/are the limiting factor in efficientAd35 vector propagation on PER.C6 cells, we have generated an Ad35adapter plasmid that does contain the E1B promoter and E1B sequences butlacks the promoter and the coding region for E1A. Hereto, the left endof wtAd35 DNA was amplified using the primers 35F1 and 35R4 (bothdescribed in Example 4) with Pwo DNA polymerase (Roche) according to themanufacturer's instructions. The 4.6 kb PCR product was purified usingthe PCR purification kit (LTI) and digested with SnaBI and ApaI enzymes.The resulting 4.2 kb fragment was then purified from gel using theQIAExII kit (Qiagen). Next, pAdApt35IP1 (Example 4) was digested withSnaBI and ApaI and the 2.6 kb vector-containing fragment was isolatedfrom gel using the GeneClean kit (BIO 101, Inc). Both isolated fragmentswere ligated to give pBr/Ad35.leftITR-pIX (FIG.16). Correctamplification during PCR was verified by a functionality test asfollows: The DNA was digested with BstBI to liberate the Ad35 insertfrom vector sequences and 4 μg of this DNA was co-transfected with 4 μgof NotI digested pWE/Ad35.pIX-rITR (Example 4) into PER.C6 cells. Thetransfected cells were passaged to T80 flasks at day 2 and again twodays later CPE had formed showing that the new pBr/Ad35.leftITR-pIXconstruct contains functional E1 sequences. The pBr/Ad35.leftITR-pIXconstruct was then further modified as follows. The DNA was digestedwith SnaBI and HindII and the 5′ HindII overhang was filled in usingKlenow enzyme. Religation of the digested DNA and transformation intocompetent cells (LTI) gave construct pBr/Ad35leftITR-pIXΔDE1A (FIG. 17).This latter construct contains the left end 4.6 kb of Ad35 except forE1A sequences between bp 450 and 1341 (numbering according to wtAd35,FIG. 5) and thus lacks the E1A promoter and most of the E1A codingsequences. pBr/Ad35.leftITR-pIXΔDE1A was then digested with BstBI and 2μg of this construct was co-transfected with 6 μmgr of NotI digestedpWE/Ad35.pIX-rITR (Example4) into PER.C6 cells. One week followingtransfection full CPE had formed in the transfected flasks.

This experiment shows that the Ad35-E1A proteins are functionallycomplemented by Ad5-E1A expression in PER.C6 cells and that at least oneof the Ad35-E1B proteins cannot be complemented by Ad5-E1 expression inPER.C6. It further shows that it is possible to make a complementingcell line for Ad35 E1-deleted viruses by expressing Ad35-E1B proteins inPER.C6. Stable expression of Ad35-E1B sequences from integrated copiesin the genome of PER.C6 cells may be driven by the E1B promoter andterminated by a heterologous poly-adenylation signal like, but notlimited to, the HBVpA. The heterologous pA signal is necessary to avoidoverlap between the E1B insert and the recombinant vector, since thenatural E1B termination is located in the pIX transcription unit thathas to be present on the adenoviral vector. Alternatively, the E1Bsequences may be driven by a heterologous promoter like, but not limitedto the human PGK promoter or by an inducible promoter like, but notlimited to the 7xtetO promoter (Gossen and Bujard, 1992). Also in thesecases the transcription termination is mediated by a heterologous pAsequence, e.g. the HBV pA. The Ad35-E1B sequences at least comprise oneof the coding regions of the E1B 21K and the E1B 55K proteins locatedbetween nucleotides 1611 and 3400 of the wt Ad35 sequence. The insertmay also include (part of the) Ad35-E1B sequences between nucleotides1550 and 1611 of the wt Ad35 sequence.

Example 7 Ad35-based Viruses Deleted for E1A and E1B-21K GenesEfficiently Propagate on Ad5 Complementing Cell Lines

The generation of Ad35-based viruses that are deleted for E1A and retainthe full E1B region is described in Example 6 of this application. Suchviruses can be generated and propagated on the Ad5 complementing cellline PER.C6. The E1B region comprises partially overlapping codingsequences for the two major proteins 21K and 55K (Bos et al., 1981).Whereas during productive wt adenoviral infection both 21K and 55K areinvolved in counteracting the apoptose-inducing effects of E1A proteins,the E1B 55K protein has been suggested to have additional functionsduring the late phase of virus infection. These include the accumulationof viral mRNAs, the control of late viral gene expression and theshutoff of most host mRNAs at the level of mRNA transport (Babiss etal., 1984, 1985; Pilder et al., 1986). A complex formed between E1B-55Kand the ORF6 protein encoded by the adenovirus early region 4 (Leppardand Shenk, 1989; Bridge and Ketner, 1990) exerts at least part of thesefunctions.

To analyze which of the E1B proteins is required for propagation ofAd35-E1A deleted recombinant viruses on PER.C6 packaging cells, the E1Bregion in construct pBr.Ad35.leftITR-pIXΔE1A (see Example 6 and FIG. 17)was further deleted. A first construct, pBr.Ad35Δ21K, retains the fullE1B-55K sequence and is deleted for E1A and E1B-21K. Hereto.pBr.Ad35.leftITR-pIXΔE1A was digested with NcoI and BspEI and the 5 KBvector fragment was isolated from agarose gel using the geneclean kit(BIO 101, Inc.) according to the manufacturer's instructions. Then a PCRfragment was generated with pBr.Ad35.leftITR-pIXΔE1A as template DNAusing the following primers:

35D21: 5′-TTA GAT CCA TGG ATC CCG CAG ACT C-3′ (SEQ ID NO: 28) and

35B3: 5′-CCT CAG CCC CAT TTC CAG-3′ (SEQ ID NO: 29).

Amplifacation was done using Pwo DNA polymerase (Roche) according tomanufacturer's recommendations with the addition of DMSO (fmalconcentration 3%) in the reaction mixture. The PCR program was asfollows: 94° C. for 2′, then 30 cycles of 94° C. for 30″, 58° C. for and72° C. for 45″and a final step at 68° C. for 8′ to ensure blunt ends.

This PCR amplifies Ad35-E1B sequences from nucl. 1908 to 2528 (sequenceAd35, FIG. 5) and introduces an NcoI site at the start codon of theE1B-55K coding sequence (bold in primer 35D21). The 620 bp PCR fragmentwas purified using the PCR purification kit (Qiagen) and then digestedwith NcoI and BspEI, purified from agarose gel as above and ligated tothe above described NcoI/BspEI digested vector fragment to givepBr.Ad35Δ21K (FIG. 18).

Since the coding regions of the 21K and 55K proteins overlap, it is onlypossible delete part of the 55K coding sequences while retaining 21K.Hereto, pBr.Ad35.leftITR-pIXΔTE1A was digested with BglII and the vectorfragment was religated to give pBr.Ad35Δ55K1 (FIG. 19). This deletionremoves E1B coding sequences from nucl. 2261 to 3330 (Ad35 sequence inFIG. 5). In this construct the N-terminal 115 amino acids are retainedand become fused to 21 additional amino acids out of the proper readingframe before a stop codon is encountered. The 21K coding region isintact in construct pBr.Ad35Δ55K1.

A third construct that has a deletion of E1A, 21K and most of the 55Ksequences was generated as follows. pBr.Ad35.leftITR-pIX (FIG. 16) wasdigested with SnaBI and MfeI (isoschizomer of MunI) and the 5′ overhangresulting from the MfeI digestion was filled in using Klenow enzyme. The4.4 kb vector fragment was isolated from gel using the geneclean kit(Bio 101, Inc.) according to the manufacturer's instructions andreligated to give construct pBr.Ad35AsM (FIG. 20). In this construct,the Ad35 sequences between nucl. 453 and 2804 are deleted thus 596 nucl.of the 3′ end of E1b-55K are retained. A further deletion of 55Ksequences was made in construct pBr.Ad35AE1A. AE1B by digestion ofpBr.Ad35.leftITR-pIX with SnaBI and BglII, Klenow treatment to fill inthe BglII cohesive ends, and religation. FIG. 21 shows a schematicrepresentation of the above mentioned constructs.

To test whether Ad35-based viruses can be generated with theseconstructs, each of the constructs was cotransfected with NotI digestedpWE.Ad35pIX-rITR (see Example 4) onto PER.C6 cells. Hereto, therespective fragments were PCR amplified using primers 35F1 and 35R4(see, Example 4). This PCR amplification was done since some of theconstructs were difficult to isolate in large enough quantities. In thisway, equal quality of the different adapter fragments was ensured. Forthe amplification Pwo DNA polymerase (Roche) was used according to themanufacturer's instructions but with DMSO (3% final concentration)addedto the PCR mixture. Of each template ˜50 ng DNA was used. The conditionsfor the PCR were as follows: 94° C. for 2′, then 5 cycles of 94° C. for30″, 48° C. for 45″ and 72° C. for 4′ followed by 25 cycles of 94° C.for 30″, 60° C. for 30″ and 72° C. for 4′ and a final step at 68° C. for8′. PCR fragments were generated from pBr.Ad35leftITR-pIX,pBr.Ad35.leftITR-pIXΔE1A, pBr.Ad35Δ21K, pBr.Ad35Δ55K1, pBr.Ad35ASM andpBr.Ad35ΔE1AΔE1B. All fragments were using the PCR purification kit(Qiagen) according to manufacturer's instructions and finalconcentrations were estimated on EtBr stained agarose gel using theEagle Eye II Still Video system and EagleSight software (Stratagene)with the SmartLadder molecular weight marker (Eurogentec) as reference.

PER.C6 cells were seeded at a density of 2.5×10⁶ cells in a T25culturing flask in DMEM containing 10% fetal calf serum (FCS) and 10 mMMgSO₄ and cultured in a humidified stove at 37° C., 10% CO₂. The nextday, 3 mg of each of the PCR fragments was cotransfected with 5 μgr NotIdigested pWE.Ad35pIX-rITR using LipofectAmine (GIBCO, Life TechnologiesInc.) according to the manufacturer's instructions. Two days after thetransfection, all cells were passed to a T80 flask and further cultured.Cultures were then monitored for the appearance of CPE. In line with theoutcome of previous experiments described in Examples 4 and 6,pBr.Ad35.leftITR-pIX and pBr.Ad35.leftITR-pIXΔE1A showed almost full CPEwithin one week following transfection. Of the fragments with differentE1B deletions only pBr.Ad35Δ21K showed CPE at the same time as the abovetwo fragments. Constructs pBr.Ad35Δ55K1, pBr.Ad35ΔSM andpEr.Ad35ΔE1AΔE1B did not give CPE at all, also not after harvesting byfreeze-thawing and re-infection of the crude lysate onto fresh PER.C6cells.

From these experiments, it can be concluded that Ad35-E1B-55K, and notE1B-21K, is necessary for generation and propagation of Ad35-basedviruses on Ad5 complementing cell lines. Therefore, Ad35-based viruseshaving a deletion of the E1A and E1B 21K genes and having the E1B-55Kgene or a functional fragment thereof, can be grown on Ad5 complementingcell lines. Alternatively, Ad35-based viruses can be grown on PER.C6cells that stably express the full E1B region or the E1B-55K gene or afunctional fragment thereof The Ad35 E1B-55K gene or functional partsthereof may be expressed from a heterologous promoter, like, but notlimited to, the human PGK promoter, the human cytomegalovirus immediateearly promoter (CMV), Rous sarcoma virus promoter, etc. and terminatedby a heterologous poly adenylation sequence (pA), like but not limitedto the hepatitis B virus poly adenylation sequence (HBVpA), the SimianVirus 40 poly adenylation sequence (SV40pA), etc. As non-limitingexamples PER.C6 cells that express the Ad35-E1B region driven by the E1Bpromoter and HBVpA, PER.C6 cells that express the Ad35-E1B region drivenby the human PGK promoter and HBVpA and PER.C6 cells that express afunctional fragment of Ad35 E1B-55K driven by the human PGK promoter andHBVpA are described below.

Generation of pIG35BL and pIG35BS

We describe the generation of two expression constructs, pIG.35BS andpIG.35BL, that both carry the Ad35-E1B genes and a neomycin selectionmarker. The two constructs differ in the length of the fragmentcontaining the E1B promoter. In 35BL the promoter fragment is longer andincludes the 3′ end of the E1A region (103 nucl. coding sequence andpA). The E1B region is terminated by the HBVpolyA, the neor genefragment on gel and isolated using the geneclean kit (BIO 101, Inc.).After religation of

pIG.35BL was made as follows. Construct pRSV.Ad35E1 (described inExample 5, FIG. 9) was digested with NruI and HindII and the protrudingends were filled in by Klenow treatment. The 7 kb vector fragment wasseparated from the smaller fragment on gel and isolated using thegeneclean kit (BIO 101, Inc.). After religation of the DNA andtransformation into competent STBL2 cells (Gibco, LTI) correct cloneswere isolated. pIG.35BL (FIG. 22) contains 273 nucl. upstream of thestart site of the E1B-21K coding region.

pIG.35BS was made in the same way as pIG.35BL except that pRSV.Ad35E1was digested with NruI and Hpal (both enzymes leave blunt ends),resulting in a shorter fragment upstream of the coding region ofE1B-21K: 97 nucleotides.

To generate Ad35-E1B expressing cells, PER.C6 cells were seeded in 10 cmdishes at 1×10⁶ cells/dish. Two days later cells were transfected withScal linearised constructs. Four dishes were transfected with 1 and fourwith 2 μg DNA (total of 16 dishes; Lipofectamine (Gibco, LTI), nocarrier DNA used) according to the manufacturer's instructions. The nextday, transfected cells received G418-containing medium (0.75 mg/ml).Control transfections using LacZ expression constructs (2 μg) werestained after 48 hrs and showed a transfection efficiency of ˜25%. Fourdays following addition of selection medium untransfected cells startedto die and again three days lateer clones were becoming visible. A weeklater, the first clones wew picked. Transfection with 1 μg resulted inless and also initially smaller clones (total ˜20 clones/dishagainst >50 clones/dish for the transfection with 2 μg DNA). Thepositive control transfection using 2 μg pcDNA3 (Invitrogen) resulted in˜50 clones.

In total, 120 clones were picked and 107 were succesfully established(55 from pIG35BS and 52 from pIG35BL).

Generation of pIG35Bneo

pIG35Bneo is an Ad35-E1B expression plasmid from which the E1B with Scaland BamHI and protruding ends were filled in using Klenow enzyme. The1070 bp fragment containing part of the Ampicilin gene and the RSVpromoter was isolated from gel using the geneclean kit (BIO 101, Inc.).Next, pRSVhbvNeo was digested with To achieve this, constructpRSVhbv.Neo (described in Example 5, FIG. 12) was digested with ScaI andBamHI and protruding ends were filled in using Klenow enzyme. The 1070were then ligated to give pRSVneo4 (FIG. 23). Construct pIG270 (FIG. 15,described in from gel using the geneclean kit (BIO 101, Inc.). Next,pRSVhbvNeo was digested with ScaI and EcoRI, blunted with Klenow and the3.2 kb fragment containing the neo gene, above and religated to givepIG270delE1A. This construct was digested with AvrII and were thenligated to give pRSVneo4 (FIG. 23). Construct pIG270 (FIG. 15, describedin Example 6) was then digested with EcoRI and NcoI and sticky ends wereblunted with Next, pRSVneo4 was digested with BglII, blunted with Klenowenzyme, above and religated to give pIG270delE1A. This construct wasdigested with AvrII and XbaI and protruding ends were filled in usingKlenow enzyme. The 2.9 kb fragment containing the hPGK promoter andAd35.E1B sequences was isolated from gel as above. Next, pRSVneo4 wasdigested with BglII, blunted with Klenow enzyme, dephosphorylated andisolated from gel. The blunted AvrII/XbaI Ad35.E1B fragment was thenligated with the above prepared pRSVneo4 vector fragment and resultingclones site at nucl 2949 in FIG. 24). was choosen and named pIG35Bneo(FIG. 24). Detailed analysis of this clone revealed coding region ofAd35.E1B-21K. Hereto, both the E1A and E1B-21K sequences are first siteat nucl 2949 in FIG. 24).

Generation of pIG35.55K

Construct pIG35.55K is similar to pIG35Bneo, however, it lacks thecoding region of Ad35.E1B-21K. Hereto, both the E1A and E1B-21Ksequences are first deleted from pIG270 as follows:

Construct pIG270 is digested with EcoRI, treated with Klenow enzyme andpurified using the PCR purification kit (Qiagen) according to themanufacturer's instructions. The recovered DNA is then digested withAgel and the ˜5 kb vector fragment was isolated from gel as above. Next,Ad35 E1B-55K sequences sre amplified by PCR on pIG270 template DNA usingthe following primers:

35D21: 5′-TTA GAT CCA TGG ATC CCG CAG ACT C-3′ (SEQ ID NO: 30) and

35B3: 5′-CCT CAG CCC CAT TTC CAG-3′ (SEQ ID NO: 31).

The conditions used for the amplification are as previously descrided.The PCR fragment is purified using the PCR purification kit (Qiagen) anddigested with NcoI. Following Klenow treatment to fill in the protrudingends, the DNA is forther sigested with AgeI and again column purified.The thus treated PRC fragment is then cloned into the above preparedEcoRI/AgeI digested vector fragment to give pIG270.ΔE1AΔ21K. The laststeps to obtain pIG35.55K (FIG. 25) are equivalent to the last stepsdescribed above for the generation of pIG35Bneo starting withpIG270.ΔE1AΔ21K instead of pIG270.ΔE1A.

pIG35.55K is then linearized with Scal and used to transfect PER.C6cells as described above. Clones that are resistent to G418 selectionare picked and analysed for their ability to complement the propagationof E1-deleted Ad35 viruses.

Example 8

New packaging cell lines for the generation and propagation ofE1-deleted Ad35-based vectors derived from primary human cells.

The complete morphological transformation of primary cells by adenovirusE1 genes is the result of the combined activities of the proteinsencoded by the E1A and E1B regions. The roles of the differint E1proteins encoded by the transformation have been studied extensively(reviewed in Zantema and van der Ed, 1995; White, 1995, 1996). Theadenovirus E1A proteins are essential for transformation of primarycells. The E1A proteins exert this effect through direct interactionwith a number of cellular proteins that are involved in regulation oftranscription. These include the pRB family of proteins, p300/CBP andTATA binding protein. In addition to this E1A increases the level of p53protein in the cells. In the absence of adenovirus E1B activity the risein p53 levels leads to the induction of apoptosis. Both proteins encodedby the E1B region counteract the induction of apoptosis although bydifferent mechanisms. E1B-21K seems to counteract apoptosis in a mannersimilar to Bcl-2 via interaction with the effector proteins downstreamin the apoptosis pathway (Han et al., 1996), whereas E1B-55K fuictionsthrough direct interaction with p53. Importantly, the molecularmechanism by which the E1B-55K proteins of Ad2 and 5 (subgroup C) andAd12 (subgroup A) function in the ability to neutralise p53 may differ.Whereas Ad5 E1B-55K binds p53 strongly and the complex localises to thecytoplasm, Ad12 E1B-55K binds p53 weakly and both proteins are localisedin the nucleus (Zantema et al., 1985; Grand et al., 1999). Bothproteins, however, inhibit the transactivation of other genes by p53(Yew and Berk, 1992).

In rodent cells, the activity of E1A together with either E1B-21K or 55Kis sufficient for full transformation although expression of both E1Bproteins together is twice as efficient (Rao et al., 1992;). In humancells however, the activity of the E1B-55K protein seems to be moreimportant given the observation that E1B-55K is indispensible for theestablishment of transformed cells (Gallimore, 1986).

Example 6 hereof describes the generation of pIG270. In this constructthe Ad35-E1 genes are expressed from the hPGK promoter and transcriptionis terminated by fragment of the Hepatitis B virus genome (Simonsen andLevinson, 1983; see also sequence described by Singer-Sam et al. (1984).The HBVpA is located in a BamHI-BglII fragment of the Hepatitis B virusgenome (Simonsen and Levinson, 1983; see also Genbank HBV-AF090841). Asmentioned before, the promoter and polyadenylation sequences of the E1expression constructs described in this invention may be derived fromother sources without departing from the invention. Also, otherfunctional fragments of the hPGK and HbVpA sequences mentioned above maybe used.

The functionality of pIG270 was shown by transformation of primary BabyRat Kidney cells (BRK). Comparison with an equivalent Ad5-E1 expressionconstruct learned that Ad35-E1 genes were less efficient in transformingthese cells. The same has been found for the E1 genes of Ad12 (Bemardset al., 1982).

It is unclear which E1 protein(s) determine(s) the difference insubgroups. In the case of Ad12, transfection studies with chimericE1A/E1B genes proteins (Bemards et al., 1982). The E1B-55K protein isshown infra to contain serotype-specific functions necessary forcomplementation of E1-deleted adenoviruses. If function, it is unlikelythat these are involved in the transformation efficiency.

Analysis of functional domains in the Ad2 or Ad5 E1B-55K proteins usinginsertion mutants have revealed that functions related to viralreplication, late protein synthesis and host protein shut-off are notconfined to specific domains but are distributed along the protein (Yewet al., 1990). Using the same set of mutants, the domains important forinteraction with p53 and E4-Orf6 were found to be more restricted. Inaddition to one common binding region (amino acids 262 to 326), p53binding was affected by mutations at aa 180 and E4-Orf6 binding wasaffected by mutations at aa 143 (Yew and Berk, 1992; Rubenwolf et al.,1997).

Altogether these results indicate that it is difficult to separate theE1B-55K functions related to transformation (p53 binding) and lateprotein synthesis (Orf6

Here is described new E1 constructs that combine the high efficiency oftransformation of one serotype with the serotype-specificcomplementation function of another serotype. These new constructs areused to transform primary human embryonic retinobladt cells and humanamniocytes.

The Generation of pIG535, pIG635 and pIG735

Construct pIG535 contains the Ad5 E1A region and E1B promoter sequenceslinked to the Ad35 E1B sequences. Hereto, pIG270 (FIG. 15; see example6) was digested with EcoRI and NcoI. The 5.3 kb vector fragment was thenisolated from gel using the geneclean kit (BIO Inc. 101) according tothe instructions of the manufacturer. Next, construct pIG.E1A.E1B (FIG.13; see example 6) was digested with EcoRI and XbaI and the resulting890 bp fragment was isolsted as above. A third fragment was generated byPRC amplification on pIG.E1A.E1B using the following primers:

5E1A-F: 5′-GAG ACG CCC GAC ATC ACC TG-3′ (SEQ ID NO: 33).

5E1B-R: 5′-CAA GCC TCC ATG GGG TCA GAT GTA AC-3′ (SEQ ID NO: 33).

The following PCR program was used: 94° C. for 2′ followed by 30 cyclesof 94° C. for 30″, 60° C. for 30″and 72° C. for 1′, and a final step at72° C. for 10′ to ensure blunt ends.

The resulting 400 bp PCR fragment was digested with XbaI and NcoI. Aftergel isolation as above, the three fragments were ligated snd transformedinto STBL-2bacteris. One colony containing all three fragments in thecorrect order was selected and designated pIG535 (FIG. 26).

Construct pIG635 contains the AdS E1A and a chimeric Ad5-Ad35 E1B regionsuch that the 21K sequence is essentially from Ad5 and linked to theAd35 E1B-55K sequences as far as not overlapping with the 21K sequences.First, part of the AdS E1 sequences are amplified by PCR usingpIG.E1A.E1B as template and the following primers:

5AK: 5′-GAG CGA AGA AAC CCA TCT GAG-3′ (SEQ ID NO: 34) and

2155R: 5′-GGT CCA GGC CGG CTC TCG G-3′ (SEQ ID NO: 35).

Amplification is accomplished with Pwo DNA polymerase (Roche) accordingto manufacturer's instruction. The 210 bp fragments is then purifiedfrom the primer sequences using the PCR purification kit (Qiagen).

A second PCR fragment is amplified from pIG270 DNA as described abovebut with the following primers:

2155F: 5′-CCG AGA GCC GGC CTG GAC-3′ (SEQ ID NO: 36) and

35F10: 5′-GCT CTA GAC CTG CAG GTT AGT CAG TTT CTT CTC CAC TG-3′ (SEQ IDNO: 37).

The 1.3 kb amplified fragment is purified as above and mixed in a 1:1molar ratio with the first PCR fragments. The mixture is then firstsubjected to a PCR reaction without the solution of primers using PwoDNA polymerase and the following program: 94° C. for 2′ and then 5cycles of 94° C. for 30′, 60° C. for 30′, 72° C. for 90″. Subsequently,primers 5AK and 35F10 are added at 0.6 μM concentration after which alast PCR amplifies a 1.5 kb fragment. Hereto, temperature was set asfollows: 94° C. for 2′, then 30 cycles of 94° C. for 30′, 60° C. for 30′and 72° C. for 90′, followed by a final step at 72° C. for 10′ to ensureblunt ends. The resulting product is purified using the PCR purificationkit (Qiagen) as above and digested with KpnI and SbfI (isoschixomer ofSse8387I). The digested DNA is then isolated from gel using thegeneclean kit (BIO Inc., 101). Construct pIG.E1A.E1B is digested withKpnl and SbfI and the vector-containing fragment is isolated from gel asabove. This fragment is ligated to the above prepared final PCR productand the ligation mixture is transformed into STBL-2 cells (Gibco, LTI)according to manufacturer's instructions. This gives construct pIG635(FIG. 27).

In construct pIG735, the border between Ad5 derived sequences and Ad35derived sequences is located more 3′ than in construct pIG635. First, aBspEI site is introduced in the Ad5 sequence of construct pIg.E1A.E1Bwithout changing the amino acid sequence. Hereto, Ad5 sequences frompIG.E1A.E1B are amplified using the following PCR primers: 5AK: (SEQ IDNO: 34) see above, and Bsp-R: 5′-GCT CTA GAC CTG CAG GGT AGC AAC AAT TCCGGA TAT TTA CAA G-3′ (SEQ ID NO: 38). Amplification is accomplishedusing Pwo DNA polymerase (Roche) according to the manufacturer'sinstruction. The following PCR program is used: 94° C. for 2′ followedby 30 cycles of 94° C. for 30′, 60° C. for 30″ and 72° C. for 30″, and afinal step at 72° C. for 10′ to ensure blunt ends. The resulting 0.6 kbfragment is purified as above and digested with KpnI and SbfI andligated to the above described KpnI/SbfI digested pIG.E1A.E1B vectorfragment. Selection of colonies after transformation of STBL-2 bacteria(Life Techn. Inc.) gives construct pIG.E1Δ55K. pIG. E1Δ55K is thendigested with SbfI and partially with BspEI. The 6.4 kb SbfI-partialBspEI digested vector fragment is then isolated from gel using thegeneclean kit (BIO 101, Inc.). Next, pIG270 is digested with BspEI andSbfI and the resulting 915 bp fragment is isolated from gel as above.This fragment is then ligated to the above prepared SbfI/partial BspEIdigested pIG.E1Δ55K vector fragment and transformed into STBL-2competent cells. This gives construct pIG735 (FIG. 28). Clones areanalysed by restriction enzyme digestion and sequencing to ensurecorrect ligation of the fragments. Constructs pIG535, pIG635 and pIG735can be used to generate complementing cell lines from primary humancells as described in Example 6.

TABLE I Elution log₁₀ [NaCl] VP/CCID50 Serotype mM VP/ml CCID50 ratio 1597 8.66 × 10¹⁰ 5.00 × 10⁷  3.2 2 574 1.04 × 10¹² 3.66 × 10¹¹ 0.4 3 1311.19 × 10¹¹ 1.28 × 10⁷  4.0 4 260 4.84 × 10¹¹ 2.50 × 10⁸  3.3 5 533 5.40× 10¹¹ 1.12 × 10¹⁰ 1.7 6 477 1.05 × 10¹² 2.14 × 10¹⁰ 1.7 7 328 1.68 ×10¹² 2.73 × 10⁹  2.4 9 379 4.99 × 10¹¹ 3.75 × 10⁷  4.1 10 387 8.32 ×10¹² 1.12 × 10⁹  3.9 12 305 3.64 × 10¹¹ 1.46 × 10⁷  4.4 13 231 4.37 ×10¹² 7.31 × 10⁸  3.8 15 443 5.33 × 10¹² 1.25 × 10⁹  3.6 16 312 1.75 ×10¹² 5.59 × 10⁸  3.5 17 478 1.39 × 10¹² 1.45 × 10⁹  3.0 19 430 8.44 ×10¹¹ 8.55 × 10⁷  4.0 20 156 1.41 × 10¹¹ 1.68 × 10⁷  3.9 21 437 3.21 ×10¹¹ 1.12 × 10⁸  3.5 22 365 1.43 × 10¹² 5.59 × 10⁷  3.4 23 132 2.33 ×10¹¹ 1.57 × 10⁷  4.2 24 405 5.12 × 10¹² 4.27 × 10⁸  4.1 25 405 7.24 ×10¹¹ 5.59 × 10⁷  4.1 26 356 1.13 × 10¹² 1.12 × 10⁸  4.0 27 342 2.00 ×10¹² 1.28 × 10⁸  4.2 28 347 2.77 × 10¹² 5.00 × 10⁷  4.7 29 386 2.78 ×10¹¹ 2.00 × 10⁷  4.1 30 409 1.33 × 10¹² 5.59 × 10⁸  3.4 31 303 8.48 ×10¹⁰ 2.19 × 10⁷  3.6 33 302 1.02 × 10¹² 1.12 × 10⁷  5.0 34 425 1.08 ×10¹² 1.63 × 10¹¹ 0.8 35 446 3.26 × 10¹² 1.25 × 10¹¹ 1.4 36 325 9.26 ×10¹² 3.62 × 10⁹  3.4 37 257 5.86 × 10¹²  2.8 × 10⁹  3.3 38 337 3.61 ×10¹² 5.59 × 10⁷  4.8 39 241 3.34 × 10¹¹ 1.17 × 10⁷  4.5 42 370 1.95 ×10¹² 1.12 × 10⁸  4.2 43 284 2.42 × 10¹² 1.81 × 10⁸  4.1 44 295 8.45 ×10¹¹ 2.00 × 10⁷  4.6 45 283 5.20 × 10¹¹ 2.99 × 10⁷  4.2 46 282 9.73 ×10¹² 2.50 × 10⁸  4.6 47 271 5.69 × 10¹¹ 3.42 × 10⁷  4.2 48 264 1.68 ×10¹² 9.56 × 10⁸  3.3 49 332 2.20 × 10¹² 8.55 × 10⁷  4.4 50 459 7.38 ×10¹² 2.80 × 10⁹  3.4 51 450 8.41 × 10¹¹ 1.88 × 10⁸  3.7

Legend to Table I:

All human adenoviruses used in the neutralization experiments wereproduced on PER.C6 cells (Fallaux et al., 1998) and purified on CsCl asdescribed in example 1. The CCID50 is shown for the 44 viruses used inthis study and reflects the HPLC column is shown. Virus particles/ml(VP/ml) were calculated from an Ad5 standard. The titer in theexperiment (CCID50) was determined on PER.C6 cells as described inExample 1 by titrations performed in parallel with the neutralizationexperiment. The CCID50 is shown for the 44 viruses used in this studyand reflects the dilution of the virus needed to obtain CPE in 50% ofthe wells after 5 days. The ratio of VP/CCID50 is depicted in log₁₀ andis a measurement of the infectively of the different batches on PER.C6cells.

TABLE II AdApt35.LacZ viruses escape neutralization by human serum.Human serum dilution Virus no serum 10x 50x 250x 1250x 6250x AdApt5.LacZ100% 0% 0% 1% 40% 80% moi: 5 VP/cell AdApt35.LacZ 100% 100% 100% 100%100% 100% 250 μl crude lysate

TABLE III The numbers of foci obtained with the different E1 expressionconstructs in BRK transformation experiments. Average of # of foci/dish:Construct 1 μgr 5 μgr Experiment 1 pIG.E1A.E1B nd 60 pIG.E1A.E1B nd 35pRSVAd35E1  0  3 pIG.Ad35.E1  3  7 Experiment 2 pIG.E1A.E1B 37 ndpIG.Ad35.E1 nd  2 Experiment 3 nd 140  pIG.Ad35.E1 nd 20 pIG270 nd 30

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39 1 14 DNA human adenovirus type 35 1 ccaataatat acct 14 2 21 DNA humanadenovirus type 35 2 aggtatatta ttgatgatgg g 21 3 18 DNA humanadenovirus 3 catcatcaat aatatacc 18 4 47 DNA Artificial Sequence Linker4 tcgatggcaa acagctatta tgggtattat gggttcgaat taattaa 47 5 47 DNAArtificial Sequence Linker 5 tcgattaatt aattcgaacc cataataccc ataatagctgtttgcca 47 6 41 DNA Artificial Sequence PCR Primer 6 ccccaattggtcgaccatca tcaataatat accttatttg g 41 7 22 DNA Artificial Sequence PCRPrimer 7 gcgaaaattg tcacttcctg tg 22 8 37 DNA Artificial Sequence Linker8 aattcggcgc gccgtcgacg atatcgatag cggccgc 37 9 37 DNA ArtificialSequence Linker 9 aattgcggcc gctatcgata tcgtcgacgg cgcgccg 37 10 49 DNAArtificial Sequence Linker 10 agctctagag gatccgttaa cgctagcgaattcaccggta ccaagctta 49 11 49 DNA Artificial Sequence Linker 11ctagtaagct tggtaccggt gaattcgcta gcgttaacgg atcctctag 49 12 44 DNAArtificial Sequence PCR Primer 12 cggaattctt aattaatcga catcatcaataatatacctt atag 44 13 33 DNA Artificial Sequence PCR Primer 13ggtggtccta ggctgacacc tacgtaaaaa cag 33 14 30 DNA Artificial SequencePCR Primer 14 tggtggagat ctggtgagta ttgggaaaac 30 15 37 DNA ArtificialSequence PCR Primer 15 cggaattctt aattaaggga aatgcaaatc tgtgagg 37 16 34DNA Artificial Sequence PCR Primer 16 cggaattcgc ggccgcggtg agtattgggaaaac 34 17 21 DNA Artificial Sequence PCR Primer 17 cgccagatcgtctacagaac a 21 18 23 DNA Artificial Sequence PCR Primer 18 gaatgctggcttcagttgta atc 23 19 42 DNA Artificial Sequence PCR Primer 19 cggaattcgcggccgcattt aaatcatcat caataatata cc 42 20 33 DNA Artificial Sequence PCRPrimer 20 ggggtaccga attctcgcta gggtatttat acc 33 21 38 DNA ArtificialSequence PCr Primer 21 gctctagacc tgcaggttag tcagtttctt ctccactg 38 2227 DNA Artificial Sequence PCR Primer 22 ggctctagag atccttcgcg ggacgtc27 23 26 DNA Artificial Sequence PCR Primer 23 ggcgaattca ctgccttccaccaagc 26 24 17 DNA Artificial Sequence Linker 24 gtgcctaggc cacgggg 1725 13 DNA Artificial Sequence Linker 25 gtggcctagg cac 13 26 20 DNAArtificial Sequence PCR Primer 26 cacctctgcc taatcatctc 20 27 27 DNAArtificial Sequence PCR Primer 27 gctctagaaa ttccactgcc ttccacc 27 28 25DNA Artificial Sequence PCR Primer 28 ttagatccat ggatcccgca gactc 25 2918 DNA Artificial Sequence PCR Primer 29 cctcagcccc atttccag 18 30 25DNA Artificial Sequence PCR Primer 30 ttagatccat ggatcccgca gactc 25 3118 DNA Artificial Sequence PCR Primer 31 cctcagcccc atttccag 18 32 20DNA Artificial Sequence PCR Primer 32 gagacgcccg acatcacctg 20 33 26 DNAArtificial Sequence PCr Primer 33 caagcctcca tggggtcaga tgtaac 26 34 21DNA Artificial Sequence PCR Primer 34 gagcgaagaa acccatctga g 21 35 19DNA Artificial Sequence PCR Primer 35 ggtccaggcc ggctctcgg 19 36 18 DNAArtificial Sequence PCR Primer 36 ccgagagccg gcctggac 18 37 38 DNAArtificial Sequence PCR Primer 37 gctctagacc tgcaggttag tcagtttcttctccactg 38 38 43 DNA Artificial Sequence PCR Primer 38 gctctagacctgcagggtag caacaattcc ggatatttac aag 43 39 34794 DNA Human AdenovirusType 35 39 catcatcaat aatatacctt atagatggaa tggtgccaat atgtaaatgaggtgatttta 60 aaaagtgtgg gccgtgtggt gattggctgt ggggttaacg gttaaaaggggcggcgcggc 120 cgtgggaaaa tgacgtttta tgggggtgga gtttttttgc aagttgtcgcgggaaatgtt 180 acgcataaaa aggcttcttt tctcacggaa ctacttagtt ttcccacggtatttaacagg 240 aaatgaggta gttttgaccg gatgcaagtg aaaattgctg attttcgcgcgaaaactgaa 300 tgaggaagtg tttttctgaa taatgtggta tttatggcag ggtggagtatttgttcaggg 360 ccaggtagac tttgacccat tacgtggagg tttcgattac cgtgttttttacctgaattt 420 ccgcgtaccg tgtcaaagtc ttctgttttt acgtaggtgt cagctgatcgctagggtatt 480 tatacctcag ggtttgtgtc aagaggccac tcttgagtgc cagcgagaagagttttctcc 540 tctgcgccgg cagtttaata ataaaaaaat gagagatttg cgatttctgcctcaggaaat 600 aatctctgct gagactggaa atgaaatatt ggagcttgtg gtgcacgccctgatgggaga 660 cgatccggag ccacctgtgc agctttttga gcctcctacg cttcaggaactgtatgattt 720 agaggtagag ggatcggagg attctaatga ggaagctgtg aatggcttttttaccgattc 780 tatgctttta gctgctaatg aaggattaga attagatccg cctttggacactttcaatac 840 tccaggggtg attgtggaaa gcggtacagg tgtaagaaaa ttacctgatttgagttccgt 900 ggactgtgat ttgcactgct atgaagacgg gtttcctccg agtgatgaggaggaccatga 960 aaaggagcag tccatgcaga ctgcagcggg tgagggagtg aaggctgccaatgttggttt 1020 tcagttggat tgcccggagc ttcctggaca tggctgtaag tcttgtgaatttcacaggaa 1080 aaatactgga gtaaaggaac tgttatgttc gctttgttat atgagaacgcactgccactt 1140 tatttacagt aagtgtgttt aagttaaaat ttaaaggaat atgctgtttttcacatgtat 1200 attgagtgtg agttttgtgc ttcttattat aggtcctgtg tctgatgctgatgaatcacc 1260 atctcctgat tctactacct cacctcctga tattcaagca cctgttcctgtggacgtgcg 1320 caagcccatt cctgtgaagc ttaagcctgg gaaacgtcca gcagtggagaaacttgagga 1380 cttgttacag ggtggggacg gacctttgga cttgagtaca cggaaacgtccaagacaata 1440 agtgttccat atccgtgttt acttaaggtg acgtcaatat ttgtgtgagagtgcaatgta 1500 ataaaaatat gttaactgtt cactggtttt tattgctttt tgggcggggactcaggtata 1560 taagtagaag cagacctgtg tggttagctc ataggagctg gctttcatccatggaggttt 1620 gggccatttt ggaagacctt aggaagacta ggcaactgtt agagagcgcttcggacggag 1680 tctccggttt ttggagattc tggttcgcta gtgaattagc tagggtagtttttaggataa 1740 aacaggacta taaacaagaa tttgaaaagt tgttggtaga ttgcccaggactttttgaag 1800 ctcttaattt gggccatcag gttcacttta aagaaaaagt tttatcagttttagactttt 1860 caaccccagg tagaactgct gctgctgtgg cttttcttac ttttatattagataaatgga 1920 tcccgcagac tcatttcagc aggggatacg ttttggattt catagccacagcattgtgga 1980 gaacatggaa ggttcgcaag atgaggacaa tcttaggtta ctggccagtgcagcctttgg 2040 gtgtagcggg aatcctgagg catccaccgg tcatgccagc ggttctggaggaggaacagc 2100 aagaggacaa cccgagagcc ggcctggacc ctccagtgga ggaggcggagtagctgactt 2160 gtctcctgaa ctgcaacggg tgcttactgg atctacgtcc actggacgggataggggcgt 2220 taagagggag agggcatcca gtggtactga tgctagatct gagttggctttaagtttaat 2280 gagtcgcaga cgtcctgaaa ccatttggtg gcatgaggtt cagaaagagggaagggatga 2340 agtttctgta ttgcaggaga aatattcact ggaacaggtg aaaacatgttggttggagcc 2400 agaggatgat tgggcggtgg ccattaaaaa ttatgccaag atagctttgaggcctgataa 2460 acagtataag atcagtagac ggattaatat ccggaatgct tgttacatatctggaaatgg 2520 ggctgaggtg gtaatagata ctcaagacaa gacagttatt agatgctgcatgatggatat 2580 gtggcctgga gtagtcggta tggaagcagt cacttttgta aatgttaagtttaggggaga 2640 tggttataat ggaatagtgt ttatggccaa taccaaactt atattgcatggttgtagctt 2700 ttttggtttc aacaatacct gtgtagatgc ctggggacag gttagtgtacgggggtgtag 2760 tttctatgcg tgttggattg ccacagctgg cagaaccaag agtcaattgtctctgaagaa 2820 atgcatattc caaagatgta acctgggcat tctgaatgaa ggcgaagcaagggtccgtca 2880 ctgcgcttct acagatactg gatgttttat tttaattaag ggaaatgccagcgtaaagca 2940 taacatgatt tgtggtgctt ccgatgagag gccttatcaa atgctcacttgtgctggtgg 3000 gcattgtaat atgctggcta ctgtgcatat tgtttcccat caacgcaaaaaatggcctgt 3060 ttttgatcac aatgtgttga ccaagtgcac catgcatgca ggtgggcgtagaggaatgtt 3120 tatgccttac cagtgtaaca tgaatcatgt gaaagtgttg ttggaaccagatgccttttc 3180 cagaatgagc ctaacaggaa tctttgacat gaacacgcaa atctggaagatcctgaggta 3240 tgatgatacg agatcgaggg tgcgcgcatg cgaatgcgga ggcaagcatgccaggttcca 3300 gccggtgtgt gtagatgtga ccgaagatct cagaccggat catttggttattgcccgcac 3360 tggagcagag ttcggatcca gtggagaaga aactgactaa ggtgagtattgggaaaactt 3420 tggggtggga ttttcagatg gacagattga gtaaaaattt gttttttctgtcttgcagct 3480 gacatgagtg gaaatgcttc ttttaagggg ggagtcttca gcccttatctgacagggcgt 3540 ctcccatcct gggcaggagt tcgtcagaat gttatgggat ctactgtggatggaagaccc 3600 gttcaacccg ccaattcttc aacgctgacc tatgctactt taagttcttcacctttggac 3660 gcagctgcag ccgctgccgc cgcctctgtc gccgctaaca ctgtgcttggaatgggttac 3720 tatggaagca tcgtggctaa ttccacttcc tctaataacc cttctacactgactcaggac 3780 aagttacttg tccttttggc ccagctggag gctttgaccc aacgtctgggtgaactttct 3840 cagcaggtgg ccgagttgcg agtacaaact gagtctgctg tcggcacggcaaagtctaaa 3900 taaaaaaaat tccagaatca atgaataaat aaacgagctt gttgttgatttaaaatcaag 3960 tgtttttatt tcatttttcg cgcacggtat gccctggacc accgatctcgatcattgaga 4020 actcggtgga ttttttccag aatcctatag aggtgggatt gaatgtttagatacatgggc 4080 attaggccgt ctttggggtg gagatagctc cattgaaggg attcatgctccggggtagtg 4140 ttgtaaatca cccagtcata acaaggtcgc agtgcatggt gttgcacaatatcttttaga 4200 agtaggctga ttgccacaga taagcccttg gtgtaggtgt ttacaaaccggttgagctgg 4260 gaggggtgca ttcgaggtga aattatgtgc attttggatt ggatttttaagttggcaata 4320 ttgccgccaa gatcccgtct tgggttcatg ttatgaagga ctaccaagacggtgtatccg 4380 gtacatttag gaaatttatc gtgcagcttg gatggaaaag cgtggaaaaatttggagaca 4440 cccttgtgtc ctccgagatt ttccatgcac tcatccatga taatagcaatggggccgtgg 4500 gcagcggcgc gggcaaacac gttccgtggg tctgacacat catagttatgttcctgagtt 4560 aaatcatcat aagccatttt aatgaatttg gggcggagcg taccagattggggtatgaat 4620 gttccttcgg gccccggagc atagttcccc tcacagattt gcatttcccaagctttcagt 4680 tctgagggtg gaatcatgtc cacctggggg gctatgaaga acaccgtttcgggggcgggg 4740 gtgattagtt gggatgatag caagtttctg agcaattgag atttgccacatccggtgggg 4800 ccataaataa ttccgattac aggttgcagg tggtagttta gggaacggcaactgccgtct 4860 tctcgaagca agggggccac ctcgttcatc atttccctta catgcatattttcccgcacc 4920 aaatccatta ggaggcgctc tcctcctagt gatagaagtt cttgtagtgaggaaaagttt 4980 ttcagcggtt ttagaccgtc agccatgggc attttggaaa gagtttgctgcaaaagttct 5040 agtctgttcc acagttcagt gatgtgttct atggcatctc gatccagcagacctcctcgt 5100 ttcgcgggtt tggacggctc ctggagtagg gtatgagacg atgggcgtccagcgctgcca 5160 gggttcggtc cttccagggt ctcagtgttc gagtcagggt tgtttccgtcacagtgaagg 5220 ggtgtgcgcc tgcttgggcg cttgccaggg tgcgcttcag actcattctgctggtggaga 5280 acttctgtcg cttggcgccc tgtatgtcgg ccaagtagca gtttaccatgagttcgtagt 5340 tgagcgcctc ggctgcgtgg cctttggcgc ggagcttacc tttggaagttttcttgcata 5400 ccgggcagta taggcatttc agcgcataca gcttgggcgc aaggaaaatggattctgggg 5460 agtatgcatc cgcgccgcag gaggcgcaaa cagtttcaca ttccaccagccaggttaaat 5520 ccggttcatt ggggtcaaaa acaagttttc cgccatattt tttgatgcgtttcttacctt 5580 tggtctccat aagttcgtgt cctcgttgag tgacaaacag gctgtccgtatctccgtaga 5640 ctgattttac aggcctcttc tccagtggag tgcctcggtc ttcttcgtacaggaactctg 5700 accactctga tacaaaggcg cgcgtccagg ccagcacaaa ggaggctatgtgggaggggt 5760 agcgatcgtt gtcaaccagg gggtccacct tttccaaagt atgcaaacacatgtcaccct 5820 cttcaacatc caggaatgtg attggcttgt aggtgtattt cacgtgacctggggtccccg 5880 ctgggggggt ataaaagggg gcggttcttt gctcttcctc actgtcttccggatcgctgt 5940 ccaggaacgt cagctgttgg ggtaggtatt ccctctcgaa ggcgggcatgacctctgcac 6000 tcaggttgtc agtttctaag aacgaggagg atttgatatt gacagtgccggttgagatgc 6060 ctttcatgag gttttcgtcc atttggtcag aaaacacaat ttttttattgtcaagtttgg 6120 tggcaaatga tccatacagg gcgttggata aaagtttggc aatggatcgcatggtttggt 6180 tcttttcctt gtccgcgcgc tctttggcgg cgatgttgag ttggacatactcgcgtgcca 6240 ggcacttcca ttcggggaag atagttgtta attcatctgg cacgattctcacttgccacc 6300 ctcgattatg caaggtaatt aaatccacac tggtggccac ctcgcctcgaaggggttcat 6360 tggtccaaca gagcctacct cctttcctag aacagaaagg gggaagtgggtctagcataa 6420 gttcatcggg agggtctgca tccatggtaa agattcccgg aagtaaatccttatcaaaat 6480 agctgatggg agtggggtca tctaaggcca tttgccattc tcgagctgccagtgcgcgct 6540 catatgggtt aaggggactg ccccagggca tgggatgggt gagagcagaggcatacatgc 6600 cacagatgtc atagacgtag atgggatcct caaagatgcc tatgtaggttggatagcatc 6660 gcccccctct gatacttgct cgcacatagt catatagttc atgtgatggcgctagcagcc 6720 ccggacccaa gttggtgcga ttgggttttt ctgttctgta gacgatctggcgaaagatgg 6780 cgtgagaatt ggaagagatg gtgggtcttt gaaaaatgtt gaaatgggcatgaggtagac 6840 ctacagagtc tctgacaaag tgggcataag attcttgaag cttggttaccagttcggcgg 6900 tgacaagtac gtctagggcg cagtagtcaa gtgtttcttg aatgatgtcataacctggtt 6960 ggtttttctt ttcccacagt tcgcggttga gaaggtattc ttcgcgatccttccagtact 7020 cttctagcgg aaacccgtct ttgtctgcac ggtaagatcc tagcatgtagaactgattaa 7080 ctgccttgta agggcagcag cccttctcta cgggtagaga gtatgcttgagcagcttttc 7140 gtagcgaagc gtgagtaagg gcaaaggtgt ctctgaccat gactttgagaaattggtatt 7200 tgaagtccat gtcgtcacag gctccctgtt cccagagttg gaagtctacccgtttcttgt 7260 aggcggggtt gggcaaagcg aaagtaacat cattgaagag aatcttaccggctctgggca 7320 taaaattgcg agtgatgcgg aaaggctgtg gtacttccgc tcgattgttgatcacctggg 7380 cagctaggac gatttcgtcg aaaccgttga tgttgtgtcc tacgatgtataattctatga 7440 aacgcggcgt gcctctgacg tgaggtagct tactgagctc atcaaaggttaggtctgtgg 7500 ggtcagataa ggcgtagtgt tcgagagccc attcgtgcag gtgaggatttgcatgtagga 7560 atgatgacca aagatctacc gccagtgctg tttgtaactg gtcccgatactgacgaaaat 7620 gccggccaat tgccattttt tctggagtga cacagtagaa ggttctggggtcttgttgcc 7680 atcgatccca cttgagttta atggctagat cgtgggccat gttgacgagacgctcttctc 7740 ctgagagttt catgaccagc atgaaaggaa ctagttgttt gccaaaggatcccatccagg 7800 tgtaagtttc cacatcgtag gtcaggaaga gtctttctgt gcgaggatgagagccgatcg 7860 ggaagaactg gatttcctgc caccagttgg aggattggct gttgatgtgatggaagtaga 7920 agtttctgcg gcgcgccgag cattcgtgtt tgtgcttgta cagacggccgcagtagtcgc 7980 agcgttgcac gggttgtatc tcgtgaatga gctgtacctg gcttcccttgacgagaaatt 8040 tcagtgggaa gccgaggcct ggcgattgta tctcgtgctc ttctatattcgctgtatcgg 8100 cctgttcatc ttctgtttcg atggtggtca tgctgacgag cccccgcgggaggcaagtcc 8160 agacctcggc gcgggagggg cggagctgaa ggacgagagc gcgcaggctggagctgtcca 8220 gagtcctgag acgctgcgga ctcaggttag taggtaggga cagaagattaacttgcatga 8280 tcttttccag ggcgtgcggg aggttcagat ggtacttgat ttccacaggttcgtttgtag 8340 agacgtcaat ggcttgcagg gttccgtgtc ctttgggcgc cactaccgtacctttgtttt 8400 ttcttttgat cggtggtggc tctcttgctt cttgcatgct cagaagcggtgacggggacg 8460 cgcgccgggc ggcagcggtt gttccggacc cgggggcatg gctggtagtggcacgtcggc 8520 gccgcgcacg ggcaggttct ggtattgcgc tctgagaaga cttgcgtgcgccaccacgcg 8580 tcgattgacg tcttgtatct gacgtctctg ggtgaaagct accggccccgtgagcttgaa 8640 cctgaaagag agttcaacag aatcaatttc ggtatcgtta acggcagcttgtctcagtat 8700 ttcttgtacg tcaccagagt tgtcctggta ggcgatctcc gccatgaactgctcgatttc 8760 ttcctcctga agatctccgc gacccgctct ttcgacggtg gccgcgaggtcattggagat 8820 acggcccatg agttgggaga atgcattcat gcccgcctcg ttccagacgcggctgtaaac 8880 cacggccccc tcggagtctc ttgcgcgcat caccacctga gcgaggttaagctccacgtg 8940 tctggtgaag accgcatagt tgcataggcg ctgaaaaagg tagttgagtgtggtggcaat 9000 gtgttcggcg acgaagaaat acatgatcca tcgtctcagc ggcatttcgctaacatcgcc 9060 cagagcttcc aagcgctcca tggcctcgta gaagtccacg gcaaaattaaaaaactggga 9120 gtttcgcgcg gacacggtca attcctcctc gagaagacgg atgagttcggctatggtggc 9180 ccgtacttcg cgttcgaagg ctcccgggat ctcttcttcc tcttctatctcttcttccac 9240 taacatctct tcttcgtctt caggcggggg cggagggggc acgcggcgacgtcgacggcg 9300 cacgggcaaa cggtcgatga atcgttcaat gacctctccg cggcggcggcgcatggtttc 9360 agtgacggcg cggccgttct cgcgcggtcg cagagtaaaa acaccgccgcgcatctcctt 9420 aaagtggtga ctgggaggtt ctccgtttgg gagggagagg gcgctgattatacattttat 9480 taattggccc gtagggactg cgcgcagaga tctgatcgtg tcaagatccacgggatctga 9540 aaacctttcg acgaaagcgt ctaaccagtc acagtcacaa ggtaggctgagtacggcttc 9600 ttgtgggcgg gggtggttat gtgttcggtc tgggtcttct gtttcttcttcatctcggga 9660 aggtgagacg atgctgctgg tgatgaaatt aaagtaggca gttctaagacggcggatggt 9720 ggcgaggagc accaggtctt tgggtccggc ttgctggata cgcaggcgattggccattcc 9780 ccaagcatta tcctgacatc tagcaagatc tttgtagtag tcttgcatgagccgttctac 9840 gggcacttct tcctcacccg ttctgccatg catacgtgtg agtccaaatccgcgcattgg 9900 ttgtaccagt gccaagtcag ctacgactct ttcggcgagg atggcttgctgtacttgggt 9960 aagggtggct tgaaagtcat caaaatccac aaagcggtgg taagcccctgtattaatggt 10020 gtaagcacag ttggccatga ctgaccagtt aactgtctgg tgaccagggcgcacgagctc 10080 ggtgtattta aggcgcgaat aggcgcgggt gtcaaagatg taatcgttgcaggtgcgcac 10140 cagatactgg taccctataa gaaaatgcgg cggtggttgg cggtagagaggccatcgttc 10200 tgtagctgga gcgccagggg cgaggtcttc caacataagg cggtgatagccgtagatgta 10260 cctggacatc caggtgattc ctgcggcggt agtagaagcc cgaggaaactcgcgtacgcg 10320 gttccaaatg ttgcgtagcg gcatgaagta gttcattgta ggcacggtttgaccagtgag 10380 gcgcgcgcag tcattgatgc tctatagaca cggagaaaat gaaagcgttcagcgactcga 10440 ctccgtagcc tggaggaacg tgaacgggtt gggtcgcggt gtaccccggttcgagacttg 10500 tactcgagcc ggccggagcc gcggctaacg tggtattggc actcccgtctcgacccagcc 10560 tacaaaaatc caggatacgg aatcgagtcg ttttgctggt ttccgaatggcagggaagtg 10620 agtcctattt tttttttttt tttgccgctc agatgcatcc cgtgctgcgacagatgcgcc 10680 cccaacaaca gcccccctcg cagcagcagc agcagcaacc acaaaaggctgtccctgcaa 10740 ctactgcaac tgccgccgtg agcggtgcgg gacagcccgc ctatgatctggacttggaag 10800 agggcgaagg actggcacgt ctaggtgcgc cttcgcccga gcggcatccgcgagttcaac 10860 tgaaaaaaga ttctcgcgag gcgtatgtgc cccaacagaa cctatttagagacagaagcg 10920 gcgaggagcc ggaggagatg cgagcttccc gctttaacgc gggtcgtgagctgcgtcacg 10980 gtttggaccg aagacgagtg ttgcgagacg aggatttcga agttgatgaagtgacaggga 11040 tcagtcctgc cagggcacac gtggctgcag ccaaccttgt atcggcttacgagcagacag 11100 taaaggaaga gcgtaacttc caaaagtctt ttaataatca tgtgcgaaccctgattgccc 11160 gcgaagaagt tacccttggt ttgatgcatt tgtgggattt gatggaagctatcattcaga 11220 accctactag caaacctctg accgcccagc tgtttctggt ggtgcaacacagcagagaca 11280 atgaggcttt cagagaggcg ctgctgaaca tcaccgaacc cgaggggagatggttgtatg 11340 atcttatcaa cattctacag agtatcatag tgcaggagcg gagcctgggcctggccgaga 11400 aggtagctgc catcaattac tcggttttga gcttgggaaa atattacgctcgcaaaatct 11460 acaagactcc atacgttccc atagacaagg aggtgaagat agatgggttctacatgcgca 11520 tgacgctcaa ggtcttgacc ctgagcgatg atcttggggt gtatcgcaatgacagaatgc 11580 atcgcgcggt tagcgccagc aggaggcgcg agttaagcga cagggaactgatgcacagtt 11640 tgcaaagagc tctgactgga gctggaaccg agggtgagaa ttacttcgacatgggagctg 11700 acttgcagtg gcagcctagt cgcagggctc tgagcgccgc gacggcaggatgtgagcttc 11760 cttacataga agaggcggat gaaggcgagg aggaagaggg cgagtacttggaagactgat 11820 ggcacaaccc gtgttttttg ctagatggaa cagcaagcac cggatcccgcaatgcgggcg 11880 gcgctgcaga gccagccgtc cggcattaac tcctcggacg attggacccaggccatgcaa 11940 cgtatcatgg cgttgacgac tcgcaacccc gaagccttta gacagcaaccccaggccaac 12000 cgtctatcgg ccatcatgga agctgtagtg ccttcccgat ctaatcccactcatgagaag 12060 gtcctggcca tcgtgaacgc gttggtggag aacaaagcta ttcgtccagatgaggccgga 12120 ctggtataca acgctctctt agaacgcgtg gctcgctaca acagtagcaatgtgcaaacc 12180 aatttggacc gtatgataac agatgtacgc gaagccgtgt ctcagcgcgaaaggttccag 12240 cgtgatgcca acctgggttc gctggtggcg ttaaatgctt tcttgagtactcagcctgct 12300 aatgtgccgc gtggtcaaca ggattatact aactttttaa gtgctttgagactgatggta 12360 tcagaagtac ctcagagcga agtgtatcag tccggtcctg attacttctttcagactagc 12420 agacagggct tgcagacggt aaatctgagc caagctttta aaaaccttaaaggtttgtgg 12480 ggagtgcatg ccccggtagg agaaagagca accgtgtcta gcttgttaactccgaactcc 12540 cgcctgttat tactgttggt agctcctttc accgacagcg gtagcatcgaccgtaattcc 12600 tatttgggtt acctactaaa cctgtatcgc gaagccatag ggcaaagtcaggtggacgag 12660 cagacctatc aagaaattac ccaagtcagt cgcgctttgg gacaggaagacactggcagt 12720 ttggaagcca ctctgaactt cttgcttacc aatcggtctc aaaagatccctcctcaatat 12780 gctcttactg cggaggagga gaggatcctt agatatgtgc agcagagcgtgggattgttt 12840 ctgatgcaag agggggcaac tccgactgca gcactggaca tgacagcgcgaaatatggag 12900 cccagcatgt atgccagtaa ccgacctttc attaacaaac tgctggactacttgcacaga 12960 gctgccgcta tgaactctga ttatttcacc aatgccatct taaacccgcactggctgccc 13020 ccacctggtt tctacacggg cgaatatgac atgcccgacc ctaatgacggatttctgtgg 13080 gacgacgtgg acagcgatgt tttttcacct ctttctgatc atcgcacgtggaaaaaggaa 13140 ggcggtgata gaatgcattc ttctgcatcg ctgtccgggg tcatgggtgctaccgcggct 13200 gagcccgagt ctgcaagtcc ttttcctagt ctaccctttt ctctacacagtgtacgtagc 13260 agcgaagtgg gtagaataag tcgcccgagt ttaatgggcg aagaggagtacctaaacgat 13320 tccttgctca gaccggcaag agaaaaaaat ttcccaaaca atggaatagaaagtttggtg 13380 gataaaatga gtagatggaa gacttatgct caggatcaca gagacgagcctgggatcatg 13440 gggactacaa gtagagcgag ccgtagacgc cagcgccatg acagacagaggggtcttgtg 13500 tgggacgatg aggattcggc cgatgatagc agcgtgttgg acttgggtgggagaggaagg 13560 ggcaacccgt ttgctcattt gcgccctcgc ttgggtggta tgttgtgaaaaaaaataaaa 13620 aagaaaaact caccaaggcc atggcgacga gcgtacgttc gttcttctttattatctgtg 13680 tctagtataa tgaggcgagt cgtgctaggc ggagcggtgg tgtatccggagggtcctcct 13740 ccttcgtacg agagcgtgat gcagcagcag caggcgacgg cggtgatgcaatccccactg 13800 gaggctccct ttgtgcctcc gcgatacctg gcacctacgg agggcagaaacagcattcgt 13860 tactcggaac tggcacctca gtacgatacc accaggttgt atctggtggacaacaagtcg 13920 gcggacattg cttctctgaa ctatcagaat gaccacagca acttcttgaccacggtggtg 13980 cagaacaatg actttacccc tacggaagcc agcacccaga ccattaactttgatgaacga 14040 tcgcggtggg gcggtcagct aaagaccatc atgcatacta acatgccaaacgtgaacgag 14100 tatatgttta gtaacaagtt caaagcgcgt gtgatggtgt ccagaaaacctcccgacggt 14160 gctgcagttg gggatactta tgatcacaag caggatattt tggaatatgagtggttcgag 14220 tttactttgc cagaaggcaa cttttcagtt actatgacta ttgatttgatgaacaatgcc 14280 atcatagata attacttgaa agtgggtaga cagaatggag tgcttgaaagtgacattggt 14340 gttaagttcg acaccaggaa cttcaagctg ggatgggatc ccgaaaccaagttgatcatg 14400 cctggagtgt atacgtatga agccttccat cctgacattg tcttactgcctggctgcgga 14460 gtggatttta ccgagagtcg tttgagcaac cttcttggta tcagaaaaaaacagccattt 14520 caagagggtt ttaagatttt gtatgaagat ttagaaggtg gtaatattccggccctcttg 14580 gatgtagatg cctatgagaa cagtaagaaa gaacaaaaag ccaaaatagaagctgctaca 14640 gctgctgcag aagctaaggc aaacatagtt gccagcgact ctacaagggttgctaacgct 14700 ggagaggtca gaggagacaa ttttgcgcca acacctgttc cgactgcagaatcattattg 14760 gccgatgtgt ctgaaggaac ggacgtgaaa ctcactattc aacctgtagaaaaagatagt 14820 aagaatagaa gctataatgt gttggaagac aaaatcaaca cagcctatcgcagttggtat 14880 ctttcgtaca attatggcga tcccgaaaaa ggagtgcgtt cctggacattgctcaccacc 14940 tcagatgtca cctgcggagc agagcaggtt tactggtcgc ttccagacatgatgaaggat 15000 cctgtcactt tccgctccac tagacaagtc agtaactacc ctgtggtgggtgcagagctt 15060 atgcccgtct tctcaaagag cttctacaac gaacaagctg tgtactcccagcagctccgc 15120 cagtccacct cgcttacgca cgtcttcaac cgctttcctg agaaccagattttaatccgt 15180 ccgccggcgc ccaccattac caccgtcagt gaaaacgttc ctgctctcacagatcacggg 15240 accctgccgt tgcgcagcag tatccgggga gtccaacgtg tgaccgttactgacgccaga 15300 cgccgcacct gtccctacgt gtacaaggca ctgggcatag tcgcaccgcgcgtcctttca 15360 agccgcactt tctaaaaaaa aaaaatgtcc attcttatct cgcccagtaataacaccggt 15420 tggggtctgc gcgctccaag caagatgtac ggaggcgcac gcaaacgttctacccaacat 15480 cccgtgcgtg ttcgcggaca ttttcgcgct ccatggggtg ccctcaagggccgcactcgc 15540 gttcgaacca ccgtcgatga tgtaatcgat caggtggttg ccgacgcccgtaattatact 15600 cctactgcgc ctacatctac tgtggatgca gttattgaca gtgtagtggctgacgctcgc 15660 aactatgctc gacgtaagag ccggcgaagg cgcattgcca gacgccaccgagctaccact 15720 gccatgcgag ccgcaagagc tctgctacga agagctagac gcgtggggcgaagagccatg 15780 cttagggcgg ccagacgtgc agcttcgggc gccagcgccg gcaggtcccgcaggcaagca 15840 gccgctgtcg cagcggcgac tattgccgac atggcccaat cgcgaagaggcaatgtatac 15900 tgggtgcgtg acgctgccac cggtcaacgt gtacccgtgc gcacccgtccccctcgcact 15960 tagaagatac tgagcagtct ccgatgttgt gtcccagcgg cgaggatgtccaagcgcaaa 16020 tacaaggaag aaatgctgca ggttatcgca cctgaagtct acggccaaccgttgaaggat 16080 gaaaaaaaac cccgcaaaat caagcgggtt aaaaaggaca aaaaagaagaggaagatggc 16140 gatgatgggc tggcggagtt tgtgcgcgag tttgccccac ggcgacgcgtgcaatggcgt 16200 gggcgcaaag ttcgacatgt gttgagacct ggaacttcgg tggtctttacacccggcgag 16260 cgttcaagcg ctacttttaa gcgttcctat gatgaggtgt acggggatgatgatattctt 16320 gagcaggcgg ctgaccgatt aggcgagttt gcttatggca agcgtagtagaataacttcc 16380 aaggatgaga cagtgtcaat acccttggat catggaaatc ccacccctagtcttaaaccg 16440 gtcactttgc agcaagtgtt acccgtaact ccgcgaacag gtgttaaacgcgaaggtgaa 16500 gatttgtatc ccactatgca actgatggta cccaaacgcc agaagttggaggacgttttg 16560 gagaaagtaa aagtggatcc agatattcaa cctgaggtta aagtgagacccattaagcag 16620 gtagcgcctg gtctgggggt acaaactgta gacattaaga ttcccactgaaagtatggaa 16680 gtgcaaactg aacccgcaaa gcctactgcc acctccactg aagtgcaaacggatccatgg 16740 atgcccatgc ctattacaac tgacgccgcc ggtcccactc gaagatcccgacgaaagtac 16800 ggtccagcaa gtctgttgat gcccaattat gttgtacacc catctattattcctactcct 16860 ggttaccgag gcactcgcta ctatcgcagc cgaaacagta cctcccgccgtcgccgcaag 16920 acacctgcaa atcgcagtcg tcgccgtaga cgcacaagca aaccgactcccggcgccctg 16980 gtgcggcaag tgtaccgcaa tggtagtgcg gaacctttga cactgccgcgtgcgcgttac 17040 catccgagta tcatcactta atcaatgttg ccgctgcctc cttgcagatatggccctcac 17100 ttgtcgcctt cgcgttccca tcactggtta ccgaggaaga aactcgcgccgtagaagagg 17160 gatgttggga cgcggaatgc gacgctacag gcgacggcgt gctatccgcaagcaattgcg 17220 gggtggtttt ttaccagcct taattccaat tatcgctgct gcaattggcgcgataccagg 17280 catagcttcc gtggcggttc aggcctcgca acgacattga cattggaaaaaaaacgtata 17340 aataaaaaaa aatacaatgg actctgacac tcctggtcct gtgactatgttttcttagag 17400 atggaagaca tcaatttttc atccttggct ccgcgacacg gcacgaagccgtacatgggc 17460 acctggagcg acatcggcac gagccaactg aacgggggcg ccttcaattggagcagtatc 17520 tggagcgggc ttaaaaattt tggctcaacc ataaaaacat acgggaacaaagcttggaac 17580 agcagtacag gacaggcgct tagaaataaa cttaaagacc agaacttccaacaaaaagta 17640 gtcgatggga tagcttccgg catcaatgga gtggtagatt tggctaaccaggctgtgcag 17700 aaaaagataa acagtcgttt ggacccgccg ccagcaaccc caggtgaaatgcaagtggag 17760 gaagaaattc ctccgccaga aaaacgaggc gacaagcgtc cgcgtcccgatttggaagag 17820 acgctggtga cgcgcgtaga tgaaccgcct tcttatgagg aagcaacgaagcttggaatg 17880 cccaccacta gaccgatagc cccaatggcc accggggtga tgaaaccttctcagttgcat 17940 cgacccgtca ccttggattt gccccctccc cctgctgcta ctgctgtacccgcttctaag 18000 cctgtcgctg ccccgaaacc agtcgccgta gccaggtcac gtcccgggggcgctcctcgt 18060 ccaaatgcgc actggcaaaa tactctgaac agcatcgtgg gtctaggcgtgcaaagtgta 18120 aaacgccgtc gctgctttta attaaatatg gagtagcgct taacttgcctatctgtgtat 18180 atgtgtcatt acacgccgtc acagcagcag aggaaaaaag gaagaggtcgtgcgtcgacg 18240 ctgagttact ttcaagatgg ccaccccatc gatgctgccc caatgggcatacatgcacat 18300 cgccggacag gatgcttcgg agtacctgag tccgggtctg gtgcagttcgcccgcgccac 18360 agacacctac ttcaatctgg gaaataagtt tagaaatccc accgtagcgccgacccacga 18420 tgtgaccacc gaccgtagcc agcggctcat gttgcgcttc gtgcccgttgaccgggagga 18480 caatacatac tcttacaaag tgcggtacac cctggccgtg ggcgacaacagagtgctgga 18540 tatggccagc acgttctttg acattagggg cgtgttggac agaggtcccagtttcaaacc 18600 ctattctggt acggcttaca actctctggc tcctaaaggc gctccaaatgcatctcaatg 18660 gattgcaaaa ggcgtaccaa ctgcagcagc cgcaggcaat ggtgaagaagaacatgaaac 18720 agaggagaaa actgctactt acacttttgc caatgctcct gtaaaagccgaggctcaaat 18780 tacaaaagag ggcttaccaa taggtttgga gatttcagct gaaaacgaatctaaacccat 18840 ctatgcagat aaactttatc agccagaacc tcaagtggga gatgaaacttggactgacct 18900 agacggaaaa accgaagagt atggaggcag ggctctaaag cctactactaacatgaaacc 18960 ctgttacggg tcctatgcga agcctactaa tttaaaaggt ggtcaggcaaaaccgaaaaa 19020 ctcggaaccg tcgagtgaaa aaattgaata tgatattgac atggaattttttgataactc 19080 atcgcaaaga acaaacttca gtcctaaaat tgtcatgtat gcagaaaatgtaggtttgga 19140 aacgccagac actcatgtag tgtacaaacc tggaacagaa gacacaagttccgaagctaa 19200 tttgggacaa cagtctatgc ccaacagacc caactacatt ggcttcagagataactttat 19260 tggactcatg tactataaca gtactggtaa catgggggtg ctggctggtcaagcgtctca 19320 gttaaatgca gtggttgact tgcaggacag aaacacagaa ctttcttaccaactcttgct 19380 tgactctctg ggcgacagaa ccagatactt tagcatgtgg aatcaggctgtggacagtta 19440 tgatcctgat gtacgtgtta ttgaaaatca tggtgtggaa gatgaacttcccaactattg 19500 ttttccactg gacggcatag gtgttccaac aaccagttac aaatcaatagttccaaatgg 19560 agaagataat aataattgga aagaacctga agtaaatgga acaagtgagatcggacaggg 19620 taatttgttt gccatggaaa ttaaccttca agccaatcta tggcgaagtttcctttattc 19680 caatgtggct ctgtatctcc cagactcgta caaatacacc ccgtccaatgtcactcttcc 19740 agaaaacaaa aacacctacg actacatgaa cgggcgggtg gtgccgccatctctagtaga 19800 cacctatgtg aacattggtg ccaggtggtc tctggatgcc atggacaatgtcaacccatt 19860 caaccaccac cgtaacgctg gcttgcgtta ccgatctatg cttctgggtaacggacgtta 19920 tgtgcctttc cacatacaag tgcctcaaaa attcttcgct gttaaaaacctgctgcttct 19980 cccaggctcc tacacttatg agtggaactt taggaaggat gtgaacatggttctacagag 20040 ttccctcggt aacgacctgc gggtagatgg cgccagcatc agtttcacgagcatcaacct 20100 ctatgctact tttttcccca tggctcacaa caccgcttcc acccttgaagccatgctgcg 20160 gaatgacacc aatgatcagt cattcaacga ctacctatct gcagctaacatgctctaccc 20220 cattcctgcc aatgcaacca atattcccat ttccattcct tctcgcaactgggcggcttt 20280 cagaggctgg tcatttacca gactgaaaac caaagaaact ccctctttggggtctggatt 20340 tgacccctac tttgtctatt ctggttctat tccctacctg gatggtaccttctacctgaa 20400 ccacactttt aagaaggttt ccatcatgtt tgactcttca gtgagctggcctggaaatga 20460 caggttacta tctcctaacg aatttgaaat aaagcgcact gtggatggcgaaggctacaa 20520 cgtagcccaa tgcaacatga ccaaagactg gttcttggta cagatgctcgccaactacaa 20580 catcggctat cagggcttct acattccaga aggatacaaa gatcgcatgtattcattttt 20640 cagaaacttc cagcccatga gcaggcaggt ggttgatgag gtcaattacaaagacttcaa 20700 ggccgtcgcc ataccctacc aacacaacaa ctctggcttt gtgggttacatggctccgac 20760 catgcgccaa ggtcaaccct atcccgctaa ctatccctat ccactcattggaacaactgc 20820 cgtaaatagt gttacgcaga aaaagttctt gtgtgacaga accatgtggcgcataccgtt 20880 ctcgagcaac ttcatgtcta tgggggccct tacagacttg ggacagaatatgctctatgc 20940 caactcagct catgctctgg acatgacctt tgaggtggat cccatggatgagcccaccct 21000 gctttatctt ctcttcgaag ttttcgacgt ggtcagagtg catcagccacaccgcggcat 21060 catcgaggca gtctacctgc gtacaccgtt ctcggccggt aacgctaccacgtaagaagc 21120 ttcttgcttc ttgcaaatag cagctgcaac catggcctgc ggatcccaaaacggctccag 21180 cgagcaagag ctcagagcca ttgtccaaga cctgggttgc ggaccctattttttgggaac 21240 ctacgataag cgcttcccgg ggttcatggc ccccgataag ctcgcctgtgccattgtaaa 21300 tacggccgga cgtgagacgg ggggagagca ctggttggct ttcggttggaacccacgttc 21360 taacacctgc tacctttttg atccttttgg attctcggat gatcgtctcaaacagattta 21420 ccagtttgaa tatgagggtc tcctgcgccg cagcgctctt gctaccaaggaccgctgtat 21480 tacgctggaa aaatctaccc agaccgtgca gggcccccgt tctgccgcctgcggactttt 21540 ctgctgcatg ttccttcacg cctttgtgca ctggcctgac cgtcccatggacggaaaccc 21600 caccatgaaa ttgctaactg gagtgccaaa caacatgctt cattctcctaaagtccagcc 21660 caccctgtgt gacaatcaaa aagcactcta ccattttctt aatacccattcgccttattt 21720 tcgctctcat cgtacacaca tcgaaagggc cactgcgttc gaccgtatggatgttcaata 21780 atgactcatg taaacaacgt gttcaataaa catcacttta tttttttacatgtatcaagg 21840 ctctggatta cttatttatt tacaagtcga atgggttctg acgagaatcagaatgacccg 21900 caggcagtga tacgttgcgg aactgatact tgggttgcca cttgaattcgggaatcacca 21960 acttgggaac cggtatatcg ggcaggatgt cactccacag ctttctggtcagctgcaaag 22020 ctccaagcag gtcaggagcc gaaatcttga aatcacaatt aggaccagtgctctgagcgc 22080 gagagttgcg gtacaccgga ttgcagcact gaaacaccat cagcgacggatgtctcacgc 22140 ttgccagcac ggtgggatct gcaatcatgc ccacatccag atcttcagcattggcaatgc 22200 tgaacggggt catcttgcag gtctgcctac ccatggcggg cacccaattaggcttgtggt 22260 tgcaatcgca gtgcaggggg atcagtatca tcttggcctg atcctgtctgattcctggat 22320 acacggctct catgaaagca tcatattgct tgaaagcctg ctgggctttactaccctcgg 22380 tataaaacat cccgcaggac ctgctcgaaa actggttagc tgcacagccggcatcattca 22440 cacagcagcg ggcgtcattg ttggctattt gcaccacact tctgccccagcggttttggg 22500 tgattttggt tcgctcggga ttctccttta aggctcgttg tccgttctcgctggccacat 22560 ccatctcgat aatctgctcc ttctgaatca taatattgcc atgcaggcacttcagcttgc 22620 cctcataatc attgcagcca tgaggccaca acgcacagcc tgtacattcccaattatggt 22680 gggcgatctg agaaaaagaa tgtatcattc cctgcagaaa tcttcccatcatcgtgctca 22740 gtgtcttgtg actagtgaaa gttaactgga tgcctcggtg ctcttcgtttacgtactggt 22800 gacagatgcg cttgtattgt tcgtgttgct caggcattag tttaaaacaggttctaagtt 22860 cgttatccag cctgtacttc tccatcagca gacacatcac ttccatgcctttctcccaag 22920 cagacaccag gggcaagcta atcggattct taacagtgca ggcagcagctcctttagcca 22980 gagggtcatc tttagcgatc ttctcaatgc ttcttttgcc atccttctcaacgatgcgca 23040 cgggcgggta gctgaaaccc actgctacaa gttgcgcctc ttctctttcttcttcgctgt 23100 cttgactgat gtcttgcatg gggatatgtt tggtcttcct tggcttctttttggggggta 23160 tcggaggagg aggactgtcg ctccgttccg gagacaggga ggattgtgacgtttcgctca 23220 ccattaccaa ctgactgtcg gtagaagaac ctgaccccac acggcgacaggtgtttttct 23280 tcgggggcag aggtggaggc gattgcgaag ggctgcggtc cgacctggaaggcggatgac 23340 tggcagaacc ccttccgcgt tcgggggtgt gctccctgtg gcggtcgcttaactgatttc 23400 cttcgcggct ggccattgtg ttctcctagg cagagaaaca acagacatggaaactcagcc 23460 attgctgtca acatcgccac gagtgccatc acatctcgtc ctcagcgacgaggaaaagga 23520 gcagagctta agcattccac cgcccagtcc tgccaccacc tctaccctagaagataagga 23580 ggtcgacgca tctcatgaca tgcagaataa aaaagcgaaa gagtctgagacagacatcga 23640 gcaagacccg ggctatgtga caccggtgga acacgaggaa gagttgaaacgctttctaga 23700 gagagaggat gaaaactgcc caaaacagcg agcagataac tatcaccaagatgctggaaa 23760 tagggatcag aacaccgact acctcatagg gcttgacggg gaagacgcgctccttaaaca 23820 tctagcaaga cagtcgctca tagtcaagga tgcattattg gacagaactgaagtgcccat 23880 cagtgtggaa gagctcagct gcgcctacga gcttaacctt ttttcacctcgtactccccc 23940 caaacgtcag ccaaacggca cctgcgagcc aaatcctcgc ttaaacttttatccagcttt 24000 tgctgtgcca gaagtactgg ctacctatca catctttttt aaaaatcaaaaaattccagt 24060 ctcctgccgc gctaatcgca cccgcgccga tgccctactc aatctgggacctggttcacg 24120 cttacctgat atagcttcct tggaagaggt tccaaagatc ttcgagggtctgggcaataa 24180 tgagactcgg gccgcaaatg ctctgcaaaa gggagaaaat ggcatggatgagcatcacag 24240 cgttctggtg gaattggaag gcgataatgc cagactcgca gtactcaagcgaagcgtcga 24300 ggtcacacac ttcgcatatc ccgctgtcaa cctgccccct aaagtcatgacggcggtcat 24360 ggaccagtta ctcattaagc gcgcaagtcc cctttcagaa gacatgcatgacccagatgc 24420 ctgtgatgag ggtaaaccag tggtcagtga tgagcagcta acccgatggctgggcaccga 24480 ctctccccgg gatttggaag agcgtcgcaa gcttatgatg gccgtggtgctggttaccgt 24540 agaactagag tgtctccgac gtttctttac cgattcagaa accttgcgcaaactcgaaga 24600 gaatctgcac tacactttta gacacggctt tgtgcggcag gcatgcaagatatctaacgt 24660 ggaactcacc aacctggttt cctacatggg tattctgcat gagaatcgcctaggacaaag 24720 cgtgctgcac agcaccctta agggggaagc ccgccgtgat tacatccgcgattgtgtcta 24780 tctctacctg tgccacacgt ggcaaaccgg catgggtgta tggcagcaatgtttagaaga 24840 acagaacttg aaagagcttg acaagctctt acagaaatct cttaaggttctgtggacagg 24900 gttcgacgag cgcaccgtcg cttccgacct ggcagacctc atcttcccagagcgtctcag 24960 ggttactttg cgaaacggat tgcctgactt tatgagccag agcatgcttaacaattttcg 25020 ctctttcatc ctggaacgct ccggtatcct gcccgccacc tgctgcgcactgccctccga 25080 ctttgtgcct ctcacctacc gcgagtgccc cccgccgcta tggagtcactgctacctgtt 25140 ccgtctggcc aactatctct cctaccactc ggatgtgatc gaggatgtgagcggagacgg 25200 cttgctggag tgccactgcc gctgcaatct gtgcacgccc caccggtccctagcttgcaa 25260 cccccagttg atgagcgaaa cccagataat aggcaccttt gaattgcaaggccccagcag 25320 ccaaggcgat gggtcttctc ctgggcaaag tttaaaactg accccgggactgtggacctc 25380 cgcctacttg cgcaagtttg ctccggaaga ttaccacccc tatgaaatcaagttctatga 25440 ggaccaatca cagcctccaa aggccgaact ttcggcttgc gtcatcacccagggggcaat 25500 tctggcccaa ttgcaagcca tccaaaaatc ccgccaagaa tttctactgaaaaagggtaa 25560 gggggtctac cttgaccccc agaccggcga ggaactcaac acaaggttccctcaggatgt 25620 cccaacgacg agaaaacaag aagttgaagg tgcagccgcc gcccccagaagatatggagg 25680 aagattggga cagtcaggca gaggaggcgg aggaggacag tctggaggacagtctggagg 25740 aagacagttt ggaggaggaa aacgaggagg cagaggaggt ggaagaagtaaccgccgaca 25800 aacagttatc ctcggctgcg gagacaagca acagcgctac catctccgctccgagtcgag 25860 gaacccggcg gcgtcccagc agtagatggg acgagaccgg acgcttcccgaacccaacca 25920 gcgcttccaa gaccggtaag aaggatcggc agggatacaa gtcctggcgggggcataaga 25980 atgccatcat ctcctgcttg catgagtgcg ggggcaacat atccttcacgcggcgctact 26040 tgctattcca ccatggggtg aactttccgc gcaatgtttt gcattactaccgtcacctcc 26100 acagccccta ctatagccag caaatcccga cagtctcgac agataaagacagcggcggcg 26160 acctccaaca gaaaaccagc agcggcagtt agaaaataca caacaagtgcagcaacagga 26220 ggattaaaga ttacagccaa cgagccagcg caaacccgag agttaagaaatcggatcttt 26280 ccaaccctgt atgccatctt ccagcagagt cggggtcaag agcaggaactgaaaataaaa 26340 aaccgatctc tgcgttcgct caccagaagt tgtttgtatc acaagagcgaagatcaactt 26400 cagcgcactc tcgaggacgc cgaggctctc ttcaacaagt actgcgcgctgactcttaaa 26460 gagtaggcag cgaccgcgct tattcaaaaa aggcgggaat tacatcatcctcgacatgag 26520 taaagaaatt cccacgcctt acatgtggag ttatcaaccc caaatgggattggcagcagg 26580 cgcctcccag gactactcca cccgcatgaa ttggctcagc gccgggccttctatgatttc 26640 tcgagttaat gatatacgcg cctaccgaaa ccaaatactt ttggaacagtcagctcttac 26700 caccacgccc cgccaacacc ttaatcccag aaattggccc gccgccctagtgtaccagga 26760 aagtcccgct cccaccactg tattacttcc tcgagacgcc caggccgaagtccaaatgac 26820 taatgcaggt gcgcagttag ctggcggctc caccctatgt cgtcacaggcctcggcataa 26880 tataaaacgc ctgatgatca gaggccgagg tatccagctc aacgacgagtcggtgagctc 26940 tccgcttggt ctacgaccag acggaatctt tcagattgcc ggctgcgggagatcttcctt 27000 cacccctcgt caggctgttc tgactttgga aagttcgtct tcgcaaccccgctcgggcgg 27060 aatcgggacc gttcaatttg tagaggagtt tactccctct gtctacttcaaccccttctc 27120 cggatctcct gggcactacc cggacgagtt cataccgaac ttcgacgcgattagcgagtc 27180 agtggacggc tacgattgat gtctggtgac gcggctgagc tatctcggctgcgacatcta 27240 gaccactgcc gccgctttcg ctgctttgcc cgggaactta ttgagttcatctacttcgaa 27300 ctccccaagg atcaccctca aggtccggcc cacggagtgc ggattactatcgaaggcaaa 27360 atagactctc gcctgcaacg aattttctcc cagcggcccg tgctgatcgagcgagaccag 27420 ggaaacacca cggtttccat ctactgcatt tgtaatcacc ccggattgcatgaaagcctt 27480 tgctgtctta tgtgtactga gtttaataaa aactgaatta agactctcctacggactgcc 27540 gcttcttcaa cccggatttt acaaccagaa gaacaaaact tttcctgtcgtccaggactc 27600 tgttaacttc acctttccta ctcacaaact agaagctcaa cgactacaccgcttttccag 27660 aagcattttc cctactaata ctactttcaa aaccggaggt gagctccacggtctccctac 27720 agaaaaccct tgggtggaag cgggccttgt agtactagga attcttgcgggtgggcttgt 27780 gattattctt tgctacctat acacaccttg cttcactttc ctagtggtgttgtggtattg 27840 gtttaaaaaa tggggcccat actagtcttg cttgttttac tttcgcttttggaaccgggt 27900 tctgccaatt acgatccatg tctagacttt gacccagaaa actgcacacttacttttgca 27960 cccgacacaa gccgcatctg tggagttctt attaagtgcg gatgggaatgcaggtccgtt 28020 gaaattacac acaataacaa aacctggaac aataccttat ccaccacatgggagccagga 28080 gttcccgagt ggtacactgt ctctgtccga ggtcctgacg gttccatccgcattagtaac 28140 aacactttca ttttttctga aatgtgcgat ctggccatgt tcatgagcaaacagtattct 28200 ctatggcctc ctagcaagga caacatcgta acgttctcca ttgcttattgcttgtgcgct 28260 tgccttctta ctgctttact gtgcgtatgc atacacctgc ttgtaaccactcgcatcaaa 28320 aacgccaata acaaagaaaa aatgccttaa cctctttctg tttacagacatggcttctct 28380 tacatctctc atatttgtca gcattgtcac tgccgctcac ggacaaacagtcgtctctat 28440 cccactagga cataattaca ctctcatagg acccccaatc acttcagaggtcatctggac 28500 caaactggga agcgttgatt actttgatat aatctgtaac aaaacaaaaccaataatagt 28560 aacttgcaac atacaaaatc ttacattgat taatgttagc aaagtttacagcggttacta 28620 ttatggttat gacagataca gtagtcaata tagaaattac ttggttcgtgttacccagtt 28680 gaaaaccacg aaaatgccaa atatggcaaa gattcgatcc gatgacaattctctagaaac 28740 ttttacatct cccaccacac ccgacgaaaa aaacatccca gattcaatgattgcaattgt 28800 tgcagcggtg gcagtggtga tggcactaat aataatatgc atgcttttatatgcttgtcg 28860 ctacaaaaag tttcatccta aaaaacaaga tctcctacta aggcttaacatttaatttct 28920 ttttatacag ccatggtttc cactaccaca ttccttatgc ttactagtctcgcaactctg 28980 acttctgctc gctcacacct cactgtaact ataggctcaa actgcacactaaaaggacct 29040 caaggtggtc atgtcttttg gtggagaata tatgacaatg gatggtttacaaaaccatgt 29100 gaccaacctg gtagattttt ctgcaacggc agagacctaa ccattatcaacgtgacagca 29160 aatgacaaag gcttctatta tggaaccgac tataaaagta gtttagattataacattatt 29220 gtactgccat ctaccactcc agcaccccgc acaactactt tctctagcagcagtgtcgct 29280 aacaatacaa tttccaatcc aacctttgcc gcgcttttaa aacgcactgtgaataattct 29340 acaacttcac atacaacaat ttccacttca acaatcagca tcatcgctgcagtgacaatt 29400 ggaatatcta ttcttgtttt taccataacc tactacgcct gctgctatagaaaagacaaa 29460 cataaaggtg atccattact tagatttgat atttaatttg ttctttttttttatttacag 29520 tatggtgaac accaatcatg gtacctagaa atttcttctt caccatactcatctgtgctt 29580 ttaatgtttg cgctactttc acagcagtag ccacagcaac cccagactgtataggagcat 29640 ttgcttccta tgcacttttt gcttttgtta cttgcatctg cgtatgtagcatagtctgcc 29700 tggttattaa ttttttccaa cttctagact ggatccttgt gcgaattgcctacctgcgcc 29760 accatcccga ataccgcaac caaaatatcg cggcacttct tagactcatctaaaaccatg 29820 caggctatac taccaatatt tttgcttcta ttgcttccct acgctgtctcaaccccagct 29880 gcctatagta ctccaccaga acaccttaga aaatgcaaat tccaacaaccgtggtcattt 29940 cttgcttgct atcgagaaaa atcagaaatc cccccaaatt taataatgattgctggaata 30000 attaatataa tctgttgcac cataatttca tttttgatat accccctatttgattttggc 30060 tggaatgctc ccaatgcaca tgatcatcca caagacccag aggaacacattcccccacaa 30120 aacatgcaac atccaatagc gctaatagat tacgaaagtg aaccacaacccccactactc 30180 cctgctatta gttacttcaa cctaaccggc ggagatgact gaaacactcaccacctccaa 30240 ttccgccgag gatctgctcg atatggacgg ccgcgtctca gaacaacgacttgcccaact 30300 acgcatccgc cagcagcagg aacgcgtggc caaagagctc agagatgtcatccaaattca 30360 ccaatgcaaa aaaggcatat tctgtttggt aaaacaagcc aagatatcctacgagatcac 30420 cgctactgac catcgcctct cttacgaact tggcccccaa cgacaaaaatttacctgcat 30480 ggtgggaatc aaccccatag ttatcaccca acaaagtgga gatactaagggttgcattca 30540 ctgctcctgc gattccatcg agtgcaccta caccctgctg aagaccctatgcggcctaag 30600 agacctgcta ccaatgaatt aaaaaaaaat gattaataaa aaatcacttacttgaaatca 30660 gcaataaggt ctctgttgaa attttctccc agcagcacct cacttccctcttcccaactc 30720 tggtattcta aaccccgttc agcggcatac tttctccata ctttaaaggggatgtcaaat 30780 tttagctcct ctcctgtacc cacaatcttc atgtctttct tcccagatgaccaagagagt 30840 ccggctcagt gactccttca accctgtcta cccctatgaa gatgaaagcacctcccaaca 30900 cccctttata aacccagggt ttatttcccc aaatggcttc acacaaagcccagacggagt 30960 tcttacttta aaatgtttaa ccccactaac aaccacaggc ggatctctacagctaaaagt 31020 gggaggggga cttacagtgg atgacactga tggtacctta caagaaaacatacgtgctac 31080 agcacccatt actaaaaata atcactctgt agaactatcc attggaaatggattagaaac 31140 tcaaaacaat aaactatgtg ccaaattggg aaatgggtta aaatttaacaacggtgacat 31200 ttgtataaag gatagtatta acaccttatg gactggaata aaccctccacctaactgtca 31260 aattgtggaa aacactaata caaatgatgg caaacttact ttagtattagtaaaaaatgg 31320 agggcttgtt aatggctacg tgtctctagt tggtgtatca gacactgtgaaccaaatgtt 31380 cacacaaaag acagcaaaca tccaattaag attatatttt gactcttctggaaatctatt 31440 aactgaggaa tcagacttaa aaattccact taaaaataaa tcttctacagcgaccagtga 31500 aactgtagcc agcagcaaag cctttatgcc aagtactaca gcttatcccttcaacaccac 31560 tactagggat agtgaaaact acattcatgg aatatgttac tacatgactagttatgatag 31620 aagtctattt cccttgaaca tttctataat gctaaacagc cgtatgatttcttccaatgt 31680 tgcctatgcc atacaatttg aatggaatct aaatgcaagt gaatctccagaaagcaacat 31740 agctacgctg accacatccc cctttttctt ttcttacatt acagaagacgacaactaaaa 31800 taaagtttaa gtgtttttat ttaaaatcac aaaattcgag tagttattttgcctccacct 31860 tcccatttga cagaatacac caatctctcc ccacgcacag ctttaaacatttggatacca 31920 ttagagatag acattgtttt agattccaca ttccaaacag tttcagagcgagccaatctg 31980 gggtcagtga tagataaaaa tccatcgcga tagtctttta aagcgctttcacagtccaac 32040 tgctgcggat gcgactccgg agtttggatc acggtcatct ggaagaagaacgatgggaat 32100 cataatccga aaacggtatc ggacgattgt gtctcatcaa acccacaagcagccgctgtc 32160 tgcgtcgctc cgtgcgactg ctgtttatgg gatcagggtc cacagtttcctgaagcatga 32220 ttttaatagc ccttaacatc aactttctgg tgcgatgcgc gcagcaacgcattctgattt 32280 cactcaaatc tttgcagtag gtacaacaca ttattacaat attgtttaataaaccataat 32340 taaaagcgct ccagccaaaa ctcatatctg atataatcgc ccctgcatgaccatcatacc 32400 aaagtttaat ataaattaaa tgacgttccc tcaaaaacac actacccacatacatgatct 32460 cttttggcat gtgcatatta acaatctgtc tgtaccatgg acaacgttggttaatcatgc 32520 aacccaatat aaccttccgg aaccacactg ccaacaccgc tcccccagccatgcattgaa 32580 gtgaaccctg ctgattacaa tgacaatgaa gaacccaatt ctctcgaccgtgaatcactt 32640 gagaatgaaa aatatctata gtggcacaac atagacataa atgcatgcatcttctcataa 32700 tttttaactc ctcaggattt agaaacatat cccagggaat aggaagctcttgcagaacag 32760 taaagctggc agaacaagga agaccacgaa cacaacttac actatgcatagtcatagtat 32820 cacaatctgg caacagcggg tggtcttcag tcatagaagc tcgggtttcattttcctcac 32880 aacgtggtaa ctgggctctg gtgtaagggt gatgtctggc gcatgatgtcgagcgtgcgc 32940 gcaaccttgt cataatggag ttgcttcctg acattctcgt attttgtatagcaaaacgcg 33000 gccctggcag aacacactct tcttcgcctt ctatcctgcc gcttagcgtgttccgtgtga 33060 tagttcaagt acagccacac tcttaagttg gtcaaaagaa tgctggcttcagttgtaatc 33120 aaaactccat cgcatctaat tgttctgagg aaatcatcca cggtagcatatgcaaatccc 33180 aaccaagcaa tgcaactgga ttgcgtttca agcaggagag gagagggaagagacggaaga 33240 accatgttaa tttttattcc aaacgatctc gcagtacttc aaattgtagatcgcgcagat 33300 ggcatctctc gcccccactg tgttggtgaa aaagcacagc taaatcaaaagaaatgcgat 33360 tttcaaggtg ctcaacggtg gcttccaaca aagcctccac gcgcacatccaagaacaaaa 33420 gaataccaaa agaaggagca ttttctaact cctcaatcat catattacattcctgcacca 33480 ttcccagata attttcagct ttccagcctt gaattattcg tgtcagttcttgtggtaaat 33540 ccaatccaca cattacaaac aggtcccgga gggcgccctc caccaccattcttaaacaca 33600 ccctcataat gacaaaatat cttgctcctg tgtcacctgt agcgaattgagaatggcaac 33660 atcaattgac atgcccttgg ctctaagttc ttctttaagt tctagttgtaaaaactctct 33720 catattatca ccaaactgct tagccagaag ccccccggga acaagagcaggggacgctac 33780 agtgcagtac aagcgcagac ctccccaatt ggctccagca aaaacaagattggaataagc 33840 atattgggaa ccaccagtaa tatcatcgaa gttgctggaa atataatcaggcagagtttc 33900 ttgtagaaat tgaataaaag aaaaatttgc caaaaaaaca ttcaaaacctctgggatgca 33960 aatgcaatag gttaccgcgc tgcgctccaa cattgttagt tttgaattagtctgcaaaaa 34020 taaaaaaaaa acaagcgtca tatcatagta gcctgacgaa caggtggataaatcagtctt 34080 tccatcacaa gacaagccac agggtctcca gctcgaccct cgtaaaacctgtcatcgtga 34140 ttaaacaaca gcaccgaaag ttcctcgcgg tgaccagcat gaataagtcttgatgaagca 34200 tacaatccag acatgttagc atcagttaag gagaaaaaac agccaacatagcctttgggt 34260 ataattatgc ttaatcgtaa gtatagcaaa gccacccctc gcggatacaaagtaaaaggc 34320 acaggagaat aaaaaatata attatttctc tgctgctgtt taggcaacgtcgcccccggt 34380 ccctctaaat acacatacaa agcctcatca gccatggctt accagagaaagtacagcggg 34440 cacacaaacc acaagctcta aagtcactct ccaacctstc cacaatatatatacacaagc 34500 cctaaactga cgtaatggga ctaaagtgta aaaaatcccg ccaaacccaacacacacccc 34560 gaaactgcgt caccagggaa aagtacagtt tcacttccgc aatcccaacaagcgtcactt 34620 cctctttctc acggtacgtc acatcccatt aacttacaac gtcattttcccacggccgcg 34680 ccgccccttt taaccgttaa ccccacagcc aatcaccaca cggcccacactttttaaaat 34740 cacctcattt acatattggc accattccat ctataaggta tattattgatgatg 34794

What is claimed is:
 1. A packaging cell lene capable of complementingrecombinant adenoviruses based on adenovirus serotype
 35. 2. A packagingcell line capable of complementing recombinant adenoviruses bases onserotypes from Subgroup B, wherein said cell line is derived fromprimary, diploid human cells, said primary, diploid human cells beingtransformed by adenovirus serotype 35 E1 sequences either operativelylinked on one DNA molecule or located on two separate DNA molecules,said sdequerces being operatively linked to regulatory seguencesenabling transcription and translation of encoded proteins.
 3. Apackaging cell line capable of complementing recombinant adenovirusesbases on serotypes from Subgroup B, wherein said cell line is derivedfrom primary, diploid human cells, said primary, diploid human cellsbeing transformed by adenovirus E1 sequences either operatively linkedon one DNA molecule or located on two separate DNA molecules, saidsequences being operatively linked to regulatory seguences enablingtranscription and translation of encoded proteins, wherein the primary,diploid human cells are transformed with chimeric E1 constructscomprising E1A and E1B sequences, wherein said chimeric E1 constructsconsist of E1 sequences of a serotype that enables efficienttransformation of primary human cells and E1B-55K sequences of anyserotype of subgroup B and E1B-21K sequences from any serotype, whichE1-deleted viruses of theat serotype, and wherein said E1B sequencescomprise E1B-21K and E1B-55K sequences to propagate chimeric subgroup Badenoviruses.
 4. The packaging cell line of claim 3 wherein the E1Aregion is derived from a subgroup C adenovirus and the E1B sequences arederived from adenovirus of subgroup B, more particular from adenovirustype
 35. 5. The packaging cell line of claim 3 wherein the E1A sequencesand the E1B-21K sequences are derived from a subgroup C adenovirus, andthe E1B-55K sequences that do not overlap with the E1B-21K sequences arederived from an adenovirus of subgroup B.
 6. The packaging cell line ofclaim 5 wherein the subgroup B adenovirus is type.
 7. The packaging cellline of claim 3 wherein all E1 sequences are derived from a subgroup Cadenovirus, except fro a part of the E1B-55K sequences that is necessaryfor serotype-specific complementation of an adenovirus subgroup B, saidsequences being derived from said adenovirus subgroup B.